Protein kinase inhibitors

ABSTRACT

The present invention is directed to novel protein kinase inhibitors comprising the chemical compound N-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-(3-hydroxypyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide, its pharmaceutically acceptable salts, enantiomers, and enantiomeric mixtures, and methods of use to treat protein kinase-mediated diseases or conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit to U.S. Non-Provisional applicationSer. No. 15/839,332 filed on Dec. 12, 2017 which claims benefit of U.S.Provisional Application No. 62/539,785 filed on Aug. 1, 2017 and to U.S.Provisional Application No. 62/433,410 filed on Dec. 13, 2016, theentire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel deuterated and non-deuteratedcyclic chemical compounds and salts thereof, to methods of using suchcompounds in treating protein kinase-mediated diseases or conditionssuch as autoimmune and cancer diseases or conditions to pharmaceuticalcompositions of said compounds, and to combination treatments of saidcompounds with co-administered therapeutic agents.

BACKGROUND OF THE INVENTION

The information provided herein is intended solely to assist theunderstanding of the reader. None of the information provided norreferences cited is admitted to be prior art to the present invention.

The identification of the molecular events that underlie the developmentof human diseases presents a major challenge in the design of improvedstrategies in the prevention, management, and cure of certain diseases(Lahiry P et al. Kinase mutations in human disease: interpretinggenotype-phenotype relationships. Nat Rev Genet 2010; 11(1):60-74).

The role of aberrantly regulated protein tyrosine kinases (PTKs) inhuman diseases is the subject of intense investigation (Lahiry id.).Protein kinases are regulators of cellular signaling, and theirfunctional dysregulation is common in carcinogenesis, autoimmunereactions, and many other disease states or conditions (Lahiry id.;Vargas L et al. Inhibitors of BTK and ITK: state of the new drugs forcancer, autoimmunity and inflammatory diseases. Scand J Immunol. 2013;78(2)130-9; Nobel M E et al. Protein kinase inhibitors: insights intodrug design from structure. Science. 2004; 303:1800-1805). The humangenome encodes over 500 protein kinases that share a catalytic domainconserved in sequence and structure but which are notably different inhow their catalysis is regulated (Manning G et al. The protein kinasecomplement of the human genome. Science. 2002; 298:1912-1934; Nobelid.). Protein kinases regulate key signal transduction cascades thatcontrol or are involved in the control of physiological functions,including cellular growth and proliferation, cell differentiation,cellular development, cell division, stress response, transcriptionregulation, aberrant mitogenesis, angiogenesis, abnormal endothelialcell-cell or cell-matrix interactions during vascular development,inflammation, Jun-N-terminal kinase (JNK) signal transduction andseveral other cellular processes (Manning id). Protein kinase inhibitorshave been established as promising drugs that inhibit overactive proteinkinases in cancer cells (Gross S et al. Targeting cancer with kinaseinhibitors. J Clin Invest. 2015; 125(5):1780-1789; Vargas id).

A partial, non-limiting list of kinases includes: ABL, ACK, ARG, BLK,BMX, BRK, BTK, CSK, DDR1, DDR2, EGFR, EPHA1, FGR, FMS, FRK, FYN(isoforma), FYN(isoform b), HCK, KIT, LCK, LYNa, PDGFRα, PDGFRβ, SRC, SRM, YES,PIK3CA/PIK3R1 (Manning id). Aberrant kinase activity has been observedin many disease states including benign and malignant proliferativeconditions as well as diseases resulting from inappropriate activationof the immune and nervous systems.

The novel compounds of this invention inhibit the activity of one ormore protein kinases and are expected to be useful in treatingkinase-related diseases or conditions.

SUMMARY OF INVENTION

The present invention concerns novel deuterated and non-deuteratedcyclic chemical compounds and salts thereof active on protein kinases ingeneral, and in particular as inhibitors of protein kinases.Additionally, methods of treating mammals with protein kinase-mediateddiseases or conditions by administering a therapeutically effectiveamount of the novel deuterated or non-deuterated cyclic chemicalcompound and/or salts thereof to such mammals in need thereof.

In one aspect, the present invention provides compounds having formulaI:

all salts, prodrugs, enantiomers, and enantiomeric mixtures thereof;

-   -   wherein    -   W₁, W₂, and W₃ are independently hydrogen or deuterium;    -   Y is carbon or nitrogen;    -   R₁ is        —Q—A        -   wherein        -   Q is a single bond directly attaching A to a ring carbon            atom, or a methylene or ethylene group connecting A to a            ring carbon atom; and        -   A is

-   -   -   -   wherein            -   Y₁ and Y₂ are independently carbon or nitrogen;            -   Z₁ and Z₂ are independently hydrogen, —(CH₂)_(n)—OR₅                where n is an integer number from 0 to 4 and R₅ is                hydrogen, lower alkyl, or lower alkenyl, with the                proviso that when n is 1 and R₅ is hydrogen, R₁ is not a                1-piperidinyl group, and that when n is 2 R₅ is                hydrogen, and R₁ is a 1-piperazinyl group, W₂ is                deuterium, and —NR₅R₆ where R₅ and R₆ are independently                hydrogen, lower alkyl, or lower alkenyl;            -   T₁ and T₂ are independently an integer number from 0 to                4 with the proviso that when T₁ or T₂ is 0, —(CH)T₁ or                —(CH₂)T₂ is a single bond, and T₁ and T₂ are not 0 at                the same time;

    -   R₂ and R₃ are independently hydrogen; halogen; alkoxyl; lower        alkyl or lower alkenyl, wherein the lower alkyl or lower alkenyl        is optionally substituted with one or more substituents selected        from —OH and alkoxyl, wherein alkoxyl is methoxy, ethoxy,        propyloxy, or tert-butoxy; or substituted heterocyclo including        —NR₅R₆, wherein R₅ and R₆ are independently hydrogen, lower        alkyl, or lower alkenyl;        and wherein the positions of R₁, R₂ and R₃ are exchangeable.

In one aspect, the present invention provides compounds having formula

all salts, prodrugs, enantiomers, and enantiomeric mixtures thereof:

-   -   wherein W₁, W₂, and W₃ are independently hydrogen or deuterium;    -   wherein Y is carbon or nitrogen;    -   wherein R₂, R₃, and R₄ are independently hydrogen; halogen;        alkoxyl; lower alkyl or lower alkenyl, wherein the lower alkyl        or lower alkenyl is optionally substituted with one or more        substituents selected from —OH and alkoxyl, wherein alkoxyl is        methoxy, ethoxy, propyloxy, or tert-butoxy; or substituted        heterocycle, wherein optionally substituted includes —NR₅R₆,        wherein R₅ and R₆ are independently hydrogen, lower alkyl, or        lower alkenyl; and    -   wherein X is independently hydrogen, —(CH₂)_(n)—OR₅ wherein n is        an integer number from 0 to 4 and R₅ is hydrogen, lower alkyl,        or lower alkenyl, or —NR₅R₆.

In one aspect, the present invention provides compounds having formula

all salts, prodrugs, enantiomers and enantiomeric mixtures thereof:

-   -   wherein W₁, W₂, and W₃ are independently hydrogen or deuterium;    -   wherein R₂, R₃, and R₄ are independently H; halogen; alkoxyl;        lower alkyl or lower alkenyl, wherein the lower alkyl or lower        alkenyl is optionally substituted with one or more substituents        selected from —OH and alkoxyl, wherein alkoxyl is methoxy,        ethoxy, propyloxy, or tert-butoxy; or substituted heterocyclo,        wherein optionally substituted includes —NR₅R₆, wherein R₅ and        R₆ are independently hydrogen, lower alkyl, or lower alkenyl.

In one aspect, the present invention provides compounds having formulaIV:

all salts, prodrugs, enantiomers and enantiomeric mixtures thereof:

-   -   wherein W₂ is hydrogen or deuterium.

In one aspect, the present invention provides compounds having formulaV:

all salts, prodrugs, enantiomers and enantiomeric mixtures thereof:

-   -   wherein W₂ hydrogen or deuterium.

Exemplary compounds include the following deuterated and non-deuteratedcyclic chemical compounds.

In one aspect, the present invention provides a compound having thestructure of compound I:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound II:

all salts and prodrugs hereof.

In one aspect, the present invention provides a compound having thestructure of compound III:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound IV:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound V:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound VI:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound VII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound VIII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound IX:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound X:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XI:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XIII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XIV:

all salts and prodrugs thereof.

In one aspect, the invention provides a method for treating a proteinkinase-mediated disease or condition in an animal or human subjectwherein the method involves administering to the subject an effectiveamount of one or more of a compound selected from formulas I, II, III,IV, and/or V, and preferably one or more of compounds I, II, III, IV, V,VI, VII, VIII, IX, X, XI, XII, XIII, and/or XIV (compounds I-XIV), andmore preferably compounds IV, V, X and/or XI.

The protein kinase mediated disease or condition is an autoimmunedisease or a cancer. Preferably the autoimmune disease may be at leastone of systemic lupus erythematosus (SLE), transplant rejection,multiple sclerosis (MS), systemic sclerosis (SSc), primary Sjögren'ssyndrome (pSS), rheumatoid arthritis (RA), and psoriasis. Preferably,the cancer is at least one of Philadelphia chromosome-positive (Ph+)chronic myeloid leukemia (CML), Philadelphia chromosome-positive acutelymphoblastic leukemia (Ph+ ALL), diffuse large B-cell lymphoma (DLBCL),chronic lymphocytic leukemia (CLL), follicular lymphoma, marginal zonelymphomas, mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia(WM), T-cell lymphomas, and multiple myeloma.

In one aspect, the invention provides a method of treating a subjectsuffering from a protein kinase-mediated disease or condition,comprising administering to the subject suffering from a proteinkinase-mediated disease or condition in combination with at least oneadditional therapeutic agent one or more of a compound selected fromformulas I, II, III, IV, and/or V, and preferably compounds I, II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and/or XIV (compoundsI-XIV), and more preferably compounds IV, V, X and/or XI.

The terms “treat” or “therapy” and like terms refer to theadministration of compounds in an amount effective to prevent,alleviate, or ameliorate one or more symptoms of a disease or condition,i.e., indication, and/or to prolong the survival of the subject beingtreated. The term “protein kinase-mediated disease or condition” refersto a disease or condition in which the biological function of a proteinkinase affects the development, course, and/or symptoms of the diseaseor condition. A protein kinase-mediated disease or condition includes adisease or condition for which modulation of protein kinase activityprovides a positive effect, i.e., one in which treatment with proteinkinase inhibitors, including compounds described herein, provides atherapeutic benefit to the subject with or at risk of the disease orcondition.

In one aspect, the invention provides for pharmaceutical compositionsthat include a therapeutically effective amount of one or more of acompound selected from formulas I, II, III, IV, and/or V, and preferablycompounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and/orXIV (compounds I-XIV), and more preferably compounds IV, V, X, and/or XIin free form or in a pharmaceutically acceptable salt form and at leastone pharmaceutically acceptable carrier, excipient, and/or diluent.

In reference to compounds of the invention a compound or group ofcompounds includes pharmaceutically acceptable salts of such compound(s)unless clearly indicated to the contrary, prodrug(s), and allstereoisomers and mixtures thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Metabolic stability of compounds in human liver microsomalincubations. Metabolic stability was determined for compounds I-XIII inincubations with human liver microsomal preparations. Incubations ofindividual compounds I-XIII at 1 μM concentrations were carried out forup to 1 hour with human liver microsomes (0.5 mg/mL) in 0.1 M phosphatebuffer containing 10 mM MgCl₂, 1 mM NADPH and 2 mM UDPGA at 37° C.Concentrations at specified times were determined by LC-MS/MS.

FIG. 2. (Upper panel) Plasma concentration versus time profiles forcompound IV and dasatinib in Sprague-Dawley rats following a single oralgavage dose of compound IV and dasatinib administered together and eachdosed at 2.5 mg/kg; (Lower panel) plasma concentration versus timeprofiles for compounds III and V in Sprague-Dawley rats following asingle oral gavage dose of compounds and V administered together andeach dosed at 2.5 mg/kg together.

FIG. 3. (Upper panel) Plasma concentration versus time profiles forcompounds X and III in Sprague-Dawley rats following a single oralgavage dose of compounds X and III administered together and each dosedat 5 mg/kg; (Lower panel) plasma concentration versus time profiles forcompound XI and dasatinib in Sprague-Dawley rats following a single oralgavage dose of compound Mend dasatinib administered together and eachdosed at 5 mg/kg together.

FIG. 4. Mean ratios of lung tissue concentration to plasma concentrationversus time for compound X and dasatinib in mice wherein compound X anddasatinib were administered together as a single oral dose and eachdosed at 5 mg/kg. (2-in-1 dosing, N=3).

FIG. 5. Mean ratios of lung tissue concentration to plasma concentrationversus time for compound XI and dasatinib in mice wherein compound XIand dasatinib were administered together as a single oral dose and eachdosed at 5 mg/kg. (2-in-1 dosing, N=3).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the following definitions apply unless clearly indicatedotherwise. By “chemical structure” or “chemical substructure” is meantany definable atom or group of atoms that constitute an individuallyidentifiable molecule, portion of a molecule, such as a substituentmoiety, a core which is optionally substituted and the like. Normally,chemical substructures of a ligand can have a role in binding of theligand to a target molecule, or can influence the three-dimensionalshape, electrostatic charge, and/or conformational properties of theligand.

The term “prodrug” is a compound that, upon in vivo administration, ismetabolized by one or more steps or processes or otherwise converted tothe biologically, pharmaceutically, or therapeutically active form ofthe compound. To produce a prodrug, the pharmaceutically active compoundis modified such that the active compound will be regenerated bymetabolic or hydrolytic processes.

The term “binds,” in connection with the interaction between a targetand a potential binding compound, indicates that the potential bindingcompound associates with the target to a statistically significantdegree as compared to association with proteins generally (i.e.,non-specific binding).

As used herein, the term “modulating” or “modulate” refers to an effectof altering a biological activity, especially a biological activityassociated with a particular biomolecule such as a protein kinase. Forexample, an agonist or antagonist of a particular biomolecule modulatesthe activity of that biomolecule, e.g., an enzyme, by either increasing(e.g., agonist, activator), or decreasing (e.g., antagonist, inhibitor)its activity. This type of activity is typically indicated in terms ofan half maximal effective concentration (EC₅₀) or half maximalinhibitory concentration (IC₅₀) for an activator or inhibitor,respectively. Additionally, inhibition activity can be expressed inpercent inhibition and/or Ki.

As used herein in connection with compounds of the invention, the term“synthesizing” and like terms means chemical synthesis from one or moreprecursor materials. Further, by “assaying” is meant the creation ofexperimental conditions and the gathering of data regarding a particularresult of the experimental conditions. For example, enzymes can beassayed based on their ability to act upon a detectable substrate. Acompound or ligand can be assayed based on its ability to bind to aparticular target molecule or molecules.

“D,” “d,” and “²H” refer to a deuterium atom, a stable isotope ofhydrogen with a mass twice that of hydrogen (atomic weight of 2.0144).Hydrogen naturally occurs as a mixture of the isotopes hydrogen (¹H),deuterium (²H or D), and tritium (³H or T). The natural abundance ofdeuterium is about 0.015%. A person skilled in the art would recognizethat all chemical compounds with a hydrogen atom actually are present asmixtures of the H and D isotopes, with about 0.015% being the deuteriumisotope. Compounds with a level of deuterium that has been enriched tobe greater than its natural abundance of 0.015% should be consideredunnatural, and as a result novel, over their non-enriched counterparts.The D in structural formulas and chemical compounds herein refers toincorporation of D in amounts greater than 0.015%.

The term “lower alkyl” is art-recognized, and includes saturatedaliphatic groups, including straight-chain alkyl groups andbranched-chain alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has about 6 or fewer carbon atoms in its backbone(e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain).

The term “lower alkenyl” refers to an unsaturated straight or branchedhydrocarbon having at least one carbon-carbon double bond, such asstraight or branched group of 2-6 carbon atoms, referred to herein asC₂-C₆ alkenyl.

The term “cycloalkyl” refers to a 3-7 membered monocyclic ring ofaliphatic groups, including C₃-C₇, that is optionally substituted withalkyl, alkenyl, alkoxyl, optionally substituted amino, halogens, cyano(—CN), or nitro (—NO₂).

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, and the like.

The terms “heterocyclo,” “heterocyclic,” or “heterocycle” refer to fullysaturated or unsaturated, including non-aromatic (i.e.,“heterocycloalkyl”) and aromatic (i.e., “heteroaryl”) cyclic groupshaving from 5 to 10 atoms with at least one heteroatom (e.g. oxygen(“O”), sulfur (“S”), or nitrogen (“N”)) in at least one carbonatom-containing ring. Each ring of the heterocyclic group may have 1, 2,3, or 4 heteroatoms. The heteroatoms nitrogen and sulfur may optionallybe oxidized and the nitrogen heteroatom may optionally be quaternized.Further, the heterocyclo may be optionally substituted with amino(—NR₅R₆), wherein R₅ and R₆ are independently hydrogen and/or loweralkyl, hydroxyl (—OH), alkoxyl, lower alkyl or lower alkenyl, whereinthe lower alkyl or lower alkenyl may be optionally substituted with —OHor alkoxyl groups.

“Halogen” refers to chloro (“Cl”), fluoro (“F”), bromo (“Br”), or iodo(“I”).

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or stereoisomeric or diastereomericmixtures thereof, including racemic mixtures (about 50:50 ratio ofenantiomers).

The term “pharmaceutically acceptable” means that the indicated materialdoes not have properties that would cause a reasonably prudent medicalpractitioner to avoid administration of the material to a patient,taking into consideration the disease or condition to be treated and therespective route of administration. For example, it is commonly requiredthat such a material be essentially sterile, e.g., for injectable.

The term “pharmaceutically acceptable salts” refers to salts that arenon-toxic in the amounts and concentrations at which they areadministered. The preparation of such salts can facilitate thepharmacological use by altering the physical characteristics of acompound without preventing it from exerting its physiological effect.

The term “pharmaceutically acceptable composition” refers to apharmaceutically active compound and one or more pharmaceuticallyacceptable carriers, excipients, and/or diluents.

The term “therapeutically effective” or “effective amount” is an amountof a preparation that alone, or together with further doses, and/or incombination with other therapeutic agents produces the desired response.This may involve halting the progression of the disease or delaying theonset of or preventing the disease or condition from occurring, althoughit may also imply only slowing of the disease or condition temporarily.

The term “protein kinase-mediated disease or condition” refers to adisease or condition in which the biological function of a proteinkinase affects the development, course, and/or symptoms of the diseaseor condition.

The term “mutants” refers to single or multiple amino acid changes in aprotein as compared to the wild-type protein amino acid sequence.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers, orsteps.

Compounds of the Invention

In one aspect, the present invention provides compounds having formulaI:

all salts, prodrugs, enantiomers, and enantiomeric mixtures thereof;

-   -   wherein    -   W₁, W₂, and W₃ are independently hydrogen or deuterium;    -   Y is carbon or nitrogen;    -   R₁ is        —Q—A        -   wherein        -   Q is a single bond directly attaching A to a ring carbon            atom, or a methylene or ethylene group connecting A to a            ring carbon atom; and        -   A is

-   -   -   -   wherein            -   Y₁ and Y₂ are independently carbon or nitrogen;            -   Z₁ and Z₂ are independently hydrogen, —(CH₂)_(n)—OR₅                where n is an integer number from 0 to 4, and R₅ is                hydrogen, lower alkyl, or lower alkenyl, with the                proviso that when n is 1 and R₅ s hydrogen, R₁ is not a                1-piperidinyl group, and that when n is 2, R₅ is                hydrogen, and R₁ is a 1-piperazinyl group, W₂ is                deuterium, and —NR₅R₆ where R₅ and R₆ are independently                hydrogen, lower alkyl, or lower alkenyl;            -   T₁ and T₂ are independently an integer number from 0 to                4 with the proviso that when T₁ or T₂ is 0, —(CH₂)T₁ or                —(CH₂)T₂ is a single bond, and T₁ and T₂ are not 0 at                the same time;

    -   R₂ and R₃ are independently hydrogen; halogen; alkoxyl; lower        alkyl or lower alkenyl, wherein the lower alkyl or lower alkenyl        is optionally substituted with one or more substituents selected        from —OH and alkoxyl, wherein alkoxyl is methoxy, ethoxy,        propyloxy, or tert-butoxy; or substituted heterocycle including        —NR₅R₆, wherein R₅ and R₆ are independently hydrogen, lower        alkyl, or lower alkenyl;        and wherein the positions of R₁, R₂ and R₃ are exchangeable.

Preferably W₂ is deuterium or hydrogen, more preferably hydrogen and W₁and W₃ are hydrogen. Y is preferably nitrogen. R₂ is preferably loweralkyl, more preferably methyl. R₃ is preferably lower alkyl or hydrogen,more preferably hydrogen. R₁ is preferably a substituted orunsubstituted saturated five or six membered nitrogen containingheterocyclo ring. The substituted or unsubstituted saturated fivemembered nitrogen containing heterocyclo ring can be substituted orunsubstituted pyrrolidin-1-yl, preferably 3-hydroxy- or3-amino-pyrrolidin-1-yl, more preferably 3-hydroxy pyrrolidin-1-yl.

In one aspect, the present invention provides compounds having formulaII:

all salts, prodrugs, enantiomers, and enantiomeric mixtures thereof:

wherein W₁, W₂, and W₃ are independently hydrogen or deuterium;

wherein Y is carbon or nitrogen;

wherein R₂, R₃, and R₄ are independently hydrogen; halogen; alkoxyl; lowalkyl or lower alkenyl, wherein the lower alkyl or lower alkenyl isoptionally substituted with one or more substituents selected from —OHand alkoxyl, wherein alkoxyl is methoxy, ethoxy, propyloxy, ortert-butoxy; or substituted heterocyclo, wherein optionally substitutedincludes —NR₅R₆, wherein R₅ and R₆ are independently hydrogen, loweralkyl, or lower alkenyl; and

wherein X is independently hydrogen, —(CH₂)_(n)—OR₆ wherein n is aninteger number from 0 to 4 and R₅ is hydrogen, lower alkyl, or loweralkenyl, or —NR₅R₆.

Preferably W₂ is deuterium or hydrogen, more preferably hydrogen, and W₁and W₃ are hydrogen. Y is preferably nitrogen. R₂ is preferably loweralkyl, more preferably methyl. R₃ is preferably lower alkyl or hydrogen,more preferably hydrogen. R₄ is preferably lower alkyl or hydrogen, morepreferably hydrogen. X is preferably hydrogen, hydroxyl or amine, morepreferably hydroxyl.

In one aspect, the present invention provides compounds having formulaIII:

all salts, prodrugs, enantiomers and enantiomeric mixtures thereof:

-   -   wherein W₁, W₂, and W₃ are independently hydrogen or deuterium;    -   wherein R₂, R₃, and R₄ are independently H; halogen; alkoxyl;        lower alkyl or lower alkenyl, wherein the lower alkyl or lower        alkenyl is optionally substituted with one or more substituents        selected from —OH and alkoxyl, wherein alkoxyl is methoxy,        ethoxy, propyloxy, or tert-butoxy; or substituted heterocyclo,        wherein optionally substituted includes —NR₅R₆, wherein R₅ and        R₆ are independently hydrogen, lower alkyl, or lower alkenyl.

Preferably W₂ is deuterium or hydrogen, more preferably hydrogen, and W₁and W₃ are hydrogen. Y is preferably nitrogen. R₂ is preferably loweralkyl, more preferably methyl. R₃ is preferably lower alkyl or hydrogen,more preferably hydrogen. R₄ is preferably lower alkyl or hydrogen, morepreferably hydrogen.

In one aspect, the present invention provides compounds having formulaIV:

all salts, prodrugs, enantiomers and enantiomeric mixtures thereof:

wherein W₂ is hydrogen or deuterium.

In one aspect, the present invention provides compounds having formulaV:

all salts, prodrugs, enantiomers and enantiomeric mixtures thereof:

wherein W₂ is hydrogen or deuterium.

Exemplary Compounds

In one aspect, the present invention provides a compound having thestructure of compound I:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound II:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound III:

all salts and prodrugs thereof.

In one aspect the present invention provides a compound having thestructure of compound IV:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound V:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound VI:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound VII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound VIII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound IX:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound X:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XI:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XIII:

all salts and prodrugs thereof.

In one aspect, the present invention provides a compound having thestructure of compound XIV:

all salts and prodrugs thereof.

Protein Kinase Targets and Indications of the Invention

Protein kinases play key roles in propagating biochemical signals indiverse biological pathways. As such, kinases represent importantcontrol points for small molecule therapeutic intervention. More than500 kinases have been described, and specific kinases have beenimplicated in a wide range of diseases or conditions. In one aspect, theinvention provides methods for treating a protein kinase-mediateddisease or condition in an animal or human subject (i.e., indications),such as without limitation, autoimmune disease hyperproliferativedisease, cancer, cardiovascular disease, inflammatory disease,neurological disease, and other diseases.

Preferably, the protein kinase-mediated disease or condition is anautoimmune disease or cancer. More preferably, the autoimmune disease isat least one of systemic lupus erythematosus (SLE), transplantrejection, multiple sclerosis (MS), systemic sclerosis (SSc), primarySjögren's syndrome (pSS), rheumatoid arthritis (RA), and psoriasis; andthe cancer is at least one of Philadelphia chromosome-positive (Ph+)chronic myeloid leukemia (CML), Philadelphia chromosome-positive acutelymphoblastic leukemia (Ph+ ALL), diffuse large B-cell lymphoma (DLBCL),chronic lymphocytic leukemia (CLL), follicular lymphoma, marginal zonelymphomas, mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia(WM), T-cell lymphomas, and multiple myeloma.

In another aspect, the invention provides a method for modulating theactivity of a protein kinase selected from the group consisting of ABL,ACK, ARG, BLK, BMX, BRK, BTK, CSK, DDR1, DDR2, EGFR, EPHA1, FGR, FMS,FRK, FYN, HCK, KIT, LCK, LYN, PDGFRα, PDGFRβ, SRC, SRM, YES,PIK3CA/PIK3R1 by administering an effective dose amount of one or morecompounds having formulas I, II, III, IV, and/or V, preferably one ormore of compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,XIII, and/or XIV (compounds I-XIV), and more preferably one or more ofcompounds IV, V, X, and/or XI. Upper and lower case for letters used inkinase nomenclature are used interchangeably in the present document.

In another aspect, the invention provides methods for treating a proteinkinase mediated disease or condition in an animal subject, wherein themethod involves administering to the subject an effective amount of acomposition including one or more compounds having formulas I, II, III,IV and/or V, preferably one or more of compounds I, II, III, IV, V, VI,VII, VIII, IX, X, XI, XII, XIII, and XIV (compounds I-XIV), and morepreferably one or more of compounds IV, V, X, and XI.

In one aspect, the invention provides methods for treating a disease orcondition mediated by a protein kinase selected from the groupconsisting of ABL, ACK, ARG, BLK, BMX, BRK, BTK, CSK, DDR1, DDR2, EGFR,EPHA1, FGR, FMS, FRK, FYN(isoform a), FYN(isoform b), HCK, KIT, LCK,LYNa, PDGFRα, PDGFRβ, SRC, SRM, YES, PIK3CA/PIK3R1 by administering aneffective amount of one or more compounds having formulas I, II, III,IV, and/or V, preferably one or more of compounds I, II, III, IV, V, VI,VII, VIII, IX, X, XI, XII, XIII, and/or XIV (compounds I-XIV), and morepreferably one or more of compounds IV, V, X, and/or XI.

A number of different assays for kinase activity can be utilized fortesting to determine active modulators and/or determining specificity ofa modulator for a particular kinase or group of kinases in addition tothe assay mentioned in the Examples below, the person of ordinary skillin the art will know and understand that other assays that can beutilized or can be modified for a particular application.

In a commonly used in vitro screen for measuring inhibition of a batteryof selected protein kinases (see Example 15) including ABL, ABL(E255K),ACK, ARG, BLK, BMX, BRK, BTK, CSK, DDR1, DDR2, EGFR, EPHA1, FGR, FMS,FRK, FYN, HCK, KIT, LCK, LYN, PDGFRα, PDGFRβ, SRC, SRM, YES, andPIK3CA/PIK3R1, compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII were found to display potent activity to inhibit BTK, BMX,ABL, ABL(E255K), SRC, ACK, ARG, BLK, DDR2, EPHA, FGR, FMS, FRK, FYN,HCK, LCK, LYN, PDGFRα, PDGFRβ, YES, and PIK3CA/PIK3R1, among others.

In the above referenced in vitro screen of the battery of proteinkinases (see Example 15), compounds I, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII displayed greater than 50% inhibition at 10 nMof BTK, BMX, ABL, ABL(E255K), SRC, ACK, ARG, BLK, DDR2, EPHA1, FGR, FMS,FRK, FYN(isoform a), HCK, LCK, LYNa, PDGFRα, PDGFRβ, YES, andPIK3CA/PIK3R1.

As a further test of biological activity; compounds of the inventionwere assayed for inhibition of cell growth using diffuse large B-celllymphoma cell line SU-DHL-4 and chronic myelogenous leukemia cell lineK-562 (see Example 16). In this cell-based assay, the IC₅₀ values forcompounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, andXIV were all less than 20 nM.

Protein kinase targets for compounds I, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, XIII, and XIV include, but are not limited to thefollowing: ABL, ACK, ARG, BLK, BMX, BRK, BTK, CSK, DDR1, DDR2, EGFR,EPHA1, FGR, FMS, FRK, FYN, HCK, KIT, LCK, LYN, PDGFRα, PDGFRβ, SRC, SRM,YES, and PIK3CA/PIK3R1.

The Tec family of kinases forms the second largest class of cytoplasmicprotein tyrosine kinases after the Src family kinases (SFKs) andconsists of five mammalian members: Btk, Bmx (bone marrow kinase on theX-chromosome, also known as Etk), Itk (IL-2 inducible T-cell kinase),Rlk (resting lymphocyte kinase, also known as Txk), and Tec (Hartkamp etal. Bruton's tyrosine kinase in chronic inflammation: frompathophysiology to therapy. Int J Interferon Cytokine Mediat Res. 2015;7: 27-34). Most of the Tec family of kinases are primarily expressed inthe hematopoietic system, although both Tec and Bmx are also expressedin stromal tissues such as liver and endothelial cells, respectively.Activation of Tec family kinases upon cell-surface receptor triggeringrequires relocalization of the protein to the plasma membrane, which ismediated by the interaction of the PH domain with the lipidphosphatidylinositol (3,4,5) P3, formed by activatedphosphatidylinositol-3 kinase. Subsequent phosphorylation by SFKs andautophosphorylation of tyrosine 223 result in the complete activation ofTec family of kinases.

BTK is the best-known member of the Tec family of kinases with BTKmutations leading to X-linked agammaglobulinemia in men and X-linkedimmunodeficiency in mice. BTK is a key regulator of B-cell development,activation, signaling, and survival (Hartkamp id). In addition, BTKplays an important role in a number of other hematopoieticcell-signaling pathways, e.g., toll-like receptor (TLR) and cytokinereceptor-mediated TNF-alpha production in macrophages, IgE receptor(FcepsilonRI) signaling in mast cells, inhibition of Fas/APO-1 apoptoticsignaling in B-lineage lymphoid cells, and collagen-stimulated plateletaggregation. BTK and other members of Tec family kinases can play acritical role in autoimmune diseases, such as systemic lupuserythematosus (SLE), multiple sclerosis (MS), type 1 diabetes (T1D),systemic sclerosis (SSc), primary Sjögren's syndrome (pSS), andrheumatoid arthritis (RA). The BTK inhibitor ibrutinib demonstrated highclinical activity in B-cell malignancies, especially in patients withchronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), andWaldenstrom's macroglobulinemia (WM). However, resistance to ibrutinibhas been demonstrated in a subgroup of patients receiving ibrutinibtreatment, mainly due to the development of BTK mutant enzyme C481S(Woyach J A et al. Resistance mechanisms for the Bruton's tyrosinekinase inhibitor ibrutinib. N Engl J Med. 2014; 370(24):2286-94).

The tyrosine kinase BMX regulates inflammation induced by TNF and othermediators appear to do so by regulating the shared TAK1-TAB complex(Gottar-Guillier M et al. The tyrosine kinase BMX is an essentialmediator of inflammatory arthritis in a kinase-independent manner. JImmunology. 2011: 186(10):6014). BMX kinase may play a role in thepathogenesis of glioblastoma, prostate, breast, and lung cancers. BMXhas also shown potential as an anti-vascular therapy in combination withradiation or as a sensitizer to chemotherapeutic agents. (Jarboe J S etal. Mini-review: bmx kinase inhibitors for cancer therapy. Recent PatAnticancer Drug Discov. 2013; 8(3):228-38).

Src family kinases (SFKs) consist of 11 nonreceptor tyrosine kinases,including Src, Fyn, Yes, Blk, Yrk, Frk (also known as Rak), Fgr, Hck,Lck, Srm, and Lyn (Sen B, Johnson F M. Regulation of SRC family kinasesin human cancers. J Signal Transduct. 2011: ID865819). Src is found inkeratinocytes, whereas Blk, Fgr, Hck, Lck, and Lyn are found primarilyin hematopoietic cells. Frk occurs chiefly in bladder, breast, brain,colon, and lymphoid cells. Src family kinases are involved inproliferation and migration responses in many cell types.

Src is a non-receptor protein tyrosine kinase that plays a multitude ofroles in cell signaling. Src is involved in the control of manyfunctions, including cell adhesion, growth, movement, anddifferentiation. Src is widely expressed in many cell types, and canhave different locations within a cell. Numerous human malignanciesdisplay increased SRC expression and activity, suggesting that SRC maybe intimately involved oncogenesis. SRC inhibitor bosutinib has beenused or the treatment of Philadelphia chromosome-positive (Ph+) chronicmyelogenous leukemia (CML), and saracatinib has been studied forpotential treatment of Alzheimer's disease and schizophrenia.

ABL is a cytoplasmic and nuclear protein tyrosine kinase that has beenimplicated in processes of cell differentiation, cell division, celladhesion, and stress response (Hantschel O. Structure, regulation,signaling, and targeting of abl kinases in cancer. Genes Cancer. 2012;3:436-46). ABL mutations are associated with cancers such as chronicmyeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and acutemyelogenous leukemia (AML). Several ABL inhibitors such as imatinib,dasatinib, and nilotinib have been used for treatment of CML, ALL, andAML, Dasatinib, a potent inhibitor of BCR-ABL is used for treatment ofnewly diagnosed Philadelphia chromosome-positive (Ph+) chronic myeloidleukemia (CML) in chronic phase, chronic, accelerated, or myeloid orlymphoid blast phase Ph+ CML with resistance or intolerance to priortherapy including imatinib, and Philadelphia chromosome-positive acutelymphoblastic leukemia (Ph+ ALL) with resistance or intolerance to priortherapy. However, dasatinib is associated with severe respiratorytoxicity such as pleural effusion and pulmonary hypertension which couldbe a result of dasatinib accumulation in lung tissue. (Quintás-CardamaA, et al. Pleural effusion in patients with chronic myelogenous leukemiatreated with dasatinib after imatinib failure. J Clin Oncol. 2007;25(25):3908-14; Guignabert C, et al. Dasatinib induces lung vasculartoxicity and predisposes to pulmonary hypertension J Clin Invest. 2016;126(9):3207-18).

LCK is a 57.9 kDa membrane-associated non-receptor tyrosine kinaseencoded by chromosome Ip34.3. The protein structure comprises an SH3 andSH2 domain. LCK inhibitors may be useful in treating acute lymphoblasticleukemia, T-cell lymphoma, lymphopenia, renal carcinoma, coloncarcinoma, severe combined immunodeficiency, multiple sclerosis,inflammatory bowel and type I diabetes.

Frk is a 58.5 kDa tyrosine kinase encoded by chromosome 6q21-q22.3. Thestructure comprises an SH2, an SH3, and a tyrosine kinase domain.Inhibition of Frk could provide means to suppress beta-cell destructionin type I diabetes. Frk inhibitors may be useful in treating acutemyeloid leukemia and type I diabetes.

Fyn is a 60.6 kDa non-receptor tyrosine kinase encoded by chromosome6q21. Fyn is involved in regulation of mast cell degranulation in asynergistic confluence of Fyn and Lyn pathways at the level of proteinkinase C and calcium regulation. Fyn inhibitors may be useful intreating Alzheimer's disease, schizophrenia, and in prevention ofmetastases, e.g., in melanoma and squamous cell carcinoma.

HCK is a 59.5 kDa tyrosine kinase encoded by chromosome 20ql 1.21. Theprotein structure comprises an SH3, an SH2, and a bipartite kinasedomain. HCK inhibitors may be useful in treating chronic myelogenousleukemia and acute lymphocytic leukemia.

Kit is a 109.9 kDa transmembrane tyrosine kinase encoded by chromosome4ql2. Kit plays an important role in the development of melanocytes,mast, germ, and hematopoietic cells. Aberrant expression and/oractivation of Kit has been implicated in a variety of pathologic states.Kit inhibitors may be useful in treating malignancies, including mastcell tumors, small cell lung cancer, testicular cancer, gastrointestinalstromal tumors (GISTs), glioblastoma, astrocytoma, neuroblastoma,carcinomas of the female genital tract, sarcomas of neuroectodermalorigin, colorectal carcinoma, carcinoma in situ, Schwann cell neoplasiaassociated with neurofibromatosis, acute myelocytic leukemia, acutelymphocytic leukemia, chronic myelogenous leukemia, mastocytosis,melanoma, and canine mast cell tumors, and inflammatory diseases,including asthma, rheumatoid arthritis, allergic rhinitis, multiplesclerosis inflammatory bowel syndrome, transplant rejection, andhypereosinophilia.

LCK is a 57.9 kDa membrane associated non-receptor tyrosine kinaseencoded by chromosome Ip34.3. The protein structure comprises an SH3 andSH2 domain. LCK inhibitors may be useful in treating acute lymphoblasticleukemia, T-cell lymphoma, lymphopenia, renal carcinoma, coloncarcinoma, severe combined immunodeficiency, multiple sclerosis,inflammatory bowel, and type I diabetes.

Platelet-derived growth factor receptors (PDGF-R) are cell surfacetyrosine kinase receptors for members of the platelet-derived growthfactor (PDGF) family. PDGF subunits-A and -B are important factorsregulating cell proliferation, cellular differentiation, cell growth anddevelopment, and many diseases including cancer. There are two forms ofthe PDGF-R, alpha and beta, each encoded by a different gene. PDGFRα isa 122.7 kDa transmembrane tyrosin kinase encoded by chromosome 4ql2(symbol: PDGFRA). PDGFRβ is a 124.0 kDa transmembrane tyrosine kinaseencoded by chromosome 5q31-q32 (symbol: PDGFRB). PDGFR inhibitors may beuseful in treating various diseases such as idiopathic hypereosinophilicsyndrome, chronic eosinophilic leukemia, glioma, gastrointestinalstromal tumors (GISTs), juvenile myelomonocytic leukemia, metastaticmedulloblastoma, atherogenesis, and restenosis.

Yes is a 60.8 kDa tyrosine kinase encoded by chromosome 18pl 1.31-pl1.21 (symbol: YESI). The structure of Yes comprises SH3 and SH2 domainsfollowed by a TK domain. The YES oncogene is homologous to the Yamaguchisarcoma virus gene, and the amino acid sequence of Yes shows a highdegree of homology with that of the SRC gene product of Roussarcomavirus. The Yes kinase is highly expressed in multiple mammalian celltypes, including neurons, spermatozoa, platelets, and epithelial cells.The target kinase Yes is amplified and overexpressed in various cancersincluding esophageal squamous cell carcinoma. Yes inhibitors may beuseful in treating cancers including esophageal squamous cell carcinoma.

In one aspect, compounds of formulas I, II, III, IV, and V, preferablycompounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, andXIV (compounds I-XIV), including salts, prodrugs, and/or isomersthereof, can be used in preparation of medicaments for the treatment ofa kinase-mediated disease or condition, in particular when the diseaseor condition is an autoimmune disease or cancer.

The amounts of compounds of formulas I, II, III, IV, and V, compounds I,II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII and XIV (compoundsI-XIV), including salts, prodrugs, and/or isomers thereof, to beadministered can be determined by standard procedures taking intoaccount factors such as the compound's IC₅₀; the biological half-life ofthe compound; the age, size, and weight of the subject; and thecondition associated with the subject. In general, routineexperimentation in clinical trials will determine specific ranges foroptimal therapeutic effect for each therapeutic agent and eachadministrative protocol and administration to specific patients will beadjusted to within effective and safe ranges depending on the patient'scondition and responsiveness to initial administration. However, theultimate administration protocol will be regulated according to thejudgment of the attending clinician considering such factors as age,gender, condition, and size of the patient. Generally, doses of activecompounds may range from about 0.01 mg/kg per day to about 1000 mg/kgper day. Compounds described herein can be administered in single ormultiple doses.

Preclinical Pharmacokinetics

In Vitro Metabolic Stability:

An in vitro metabolic stability study in human liver microsomes wasconducted for compounds I through XIII and dasatinib (see Example 17 forexperimental conditions). Results from this study are shown in FIG. 1and table 1.

Compounds I, IV, V, VI, VII, VIII, X, XI, XII and XIII displayedsignificantly greater stability as compared to dasatinib. The in vitrot_(1/2) for compounds I, IV, V, VI, VII, VIII, X, XI, XII, and XIII wereall >59 min compared to 16 min for dasatinib. This significant increasein metabolic stability for compounds I, IV, V, VI, VII, VIII, X, XI,XII, and XIII was unexpected. Longer in vitro metabolic stabilityhalf-life (t_(1/2)) is an indicator of longer in vivo human plasmat_(1/2) for these compounds compared to dasatinb. Therefore, thesecompounds are expected to have a more favorable pharmacokinetic profilecompared to dasatinib, specifically longer t_(1/2), longer duration ofaction, less first pass effect, and higher oral bioavailability. Theintrinsic clearances for compounds I, IV, V, VI, VII, VIII, X, XI, XII,and XIII were less than 24 μL/min/mg compared to 88 μL/min/mg fordastatinib.

Surprisingly compound III, a deuterium labeled analog of dasatinib, wasfound to have similar metabolic stability to dasatinib (FIG. 1, table1). This was an unexpected finding given the reported in vitrometabolism of dasatinib in human liver microsomes where hydroxylation atthe 4-position of the 2-chloro-6-methylphenyl ring is a route ofoxidative metabolism (Christopher L J et al. Biotransformation of[14C]dasatinib: in vitro studies in rat, monkey, and human anddisposition after administration to rats and monkey. Drug Metab. Dispos.2007; 36(7):1341-1356).

Compounds I, IV, V, VI, VII, VIII, X, XI, XII, and XIII, showedunexpectedly significantly increased metabolic stability in human livermicrosomes compared to dasatinib, and deuterium substitution results inadditional unexpected results as compared to dasatinib.

TABLE 1 In vitro t_(1/2) intrinsic clearance (CLint) of Compounds inhuman liver microsomal incubations* In vitro t_(1/2) (min) CLint(μL/min/mg) Dasatinib 16 88 Compound I 281 5 Compound II 32 44 CompoundIII 24 58 Compound IV 88 16 Compound V 67 21 Compound VI 403 3 CompoundVII 404 3 Compound VIII >500 <3 Compound IX 25 56 Compound X 87 16Compound XI 59 24 Compound XII 448 3 Compound XIII 261 5 *CompoundI-XIII (1 μM) were incubated with human liver microsomes (0.5 mg/mL) in0.1M phosphate buffer containing 10 mM MgCl₂, 1 mM NADPH and 2 mM UDPGAat 37° C. for various time points through 60 min. The concentrations ofremaining test compounds at the various time points were determined byLC-MS/MS.

In Vivo Pharmacokinetics:

The in vivo pharmacokinetic profiles of compounds III, IV, V, X, XI, anddasatinib were investigated in Sprague Dawley rats following oral andintravenous administration using the technique of 2-in-1 dosing (seeExample 18 for experimental conditions). Compound IV was dosed withdasatinib, compound V was dosed with compound III, compound X was dosedwith dasatinib, and compound XI was dosed with compound III. Results areshown in FIGS. 2 and 3 and tables 2 and 3.

TABLE 2 Pharmacokinetic parameters of compounds III, IV, V and dasatinibin Sprague-Dawley rats following a single intravenous or oral doseadministration (Example 18). Compound IV Dasatinib Compound V CompoundIII intravenous PO intravenous PO intravenous PO intravenous PO Dose 12.5 1 2.5 1 2.5 1 2.5 (mg/kg) C_(max) N/A 82 ± 23 N/A 15 ± 5  N/A 35 ±19 N/A 9 ± 6 (ng/mL) T_(max) (hr) N/A 5.5 ± 4.3 N/A 5.5 ± 4.3 N/A 5.5 ±4.3 N/A 5.5 ± 4.3 AUC_(last) 1877 ± 152 891 ± 341 783 ± 54 193 ± 86 1960 ± 214 524 ± 191 835 ± 114 139 ± 69  (ng/mL*hr) AUC_(inf) 1879 ± 153892 ± 341 784 ± 54 196 ± 85  1961 ± 214 539 ± 188 835 ± 114 144 ± 68 (ng/mL*hr) t_(1/2) (hr)  1.4 ± 0.1 2.3 ± 0.2 1.5 ± 0  3.5 ± 0.5  1.4 ±0.1 4.4 ± 2.8 1.6 ± 0.1 4.7 ± 2.2 CL 535 ± 45 N/A 1279 ± 88  N/A 514 ±54 N/A 1211 ± 157  N/A (mL/hr/kg) V (L/kg)  1 ± 0 N/A  3 ± 0 N/A  1 ± 0N/A 3 ± 0 N/A F (%) N/A 18.9 ± 6.7  N/A 9.8 ± 3.6 N/A 11.1 ± 4.3  N/A7.2 ± 4.2 Cmax—plasma maximum concentration following oral dosing;T_(max)—time to maximum plasma concentration following oral dosing;AUC_(last)—area under the plasma concentration versus time curve to thelast detectable concentration; AUC_(inf)—area under the plasmaconcentration versus time curve extrapolated to time infinity;t_(1/2)—plasma concentration half-life; CL—plasma clearance; V—volume ofdistribution; F (%)—percent oral bioavailability as determined byAUC_(inf) (oral) versus AUC_(inf) (intravenous) dose normalized.

TABLE 3 Pharmacokinetic parameters of compounds III, X, XI, anddasatinib in Sprague-Dawley rats following a single intravenous or oraldose. Compound X Dasatinib Compound XI Compound III intravenous POintravenous PO intravenous PO intravenous PO Dose 1 5 1 5 1 5 1 5(mg/kg) C_(max) N/A  93 ± 101 N/A 7 ± 7 N/A 102 ± 86 N/A 11 ± 11 (ng/mL)T_(max) (hr) N/A 0.5 ± 0  N/A 0.5 ± 0  N/A 0.5 ± 0  N/A 0.5 ± 0 AUC_(last) 2519 ± 446 1122 ± 369 1013 ± 68 93 ± 23 2480 ± 243 1420 ± 343916 ± 161 124 ± 26  (ng/mL*hr) AUC_(inf) 2520 ± 446 1204 ± 349 1014 ± 6897 ± 22 2481 ± 243 1495 ± 273 917 ± 161 137 ± 20  (ng/mL*hr) t½ (hr) 1.6 ± 0.2  6.8 ± 4.1  1.7 ± 0.3 5.9 ± 1.5  1.6 ± 0.2   6 ± 3.6 1.7 ±0.2 8.2 ± 4.1 CL 405 ± 66 N/A  989 ± 64 N/A 406 ± 42 N/A 1113 ± 193  N/A(mL/hr/kg) V (L/kg)  1 ± 0 N/A  2 ± 0 N/A  1 ± 0 N/A 3 ± 0 N/A F (%) N/A18.9 ± 2.2 N/A 3.9 ± 1  N/A  24 ± 3.1 N/A  6 ± 0.9 Cmax—plasma maximumconcentration following oral dosing; T_(max)—time to maximum plasmaconcentration following oral dosing; AUC_(last)—area under the plasmaconcentration versus time curve to the last detectable concentration;AUC_(inf)—area under the plasma concentration versus time curveextrapolated to time infinity; t_(1/2)—plasma concentration half-life;CL—plasma clearance; V—volume of distribution; F (%)—percent oralbioavailability as determined by AUC_(inf) (oral) versus AUC_(inf)(intravenous) dose normalized.

Following oral administrations, compounds IV, V, X, and XI showedsurprisingly significantly higher Cmax values of 82±13, 35±19, 93±101and 102±86 ng/mL, respectively, as compared to dasatinib where Cmax was15±5 and 7±7 ng/mL and the deuterium analog of dasatinib, compound III,where Cmax was 9±6 and 11±11 ng/mL. The oral bioavailability ofcompounds IV, V, X, and XI was 18.9±6.7, 11.1±4.3, 18.9±2, and 24±33.1percent bioavailability (F) compared to dasatinib oral bioavailabilityof 3.9±1 and 9.8±3.6 percent bioavailability, and compound III whereoral bioavailability was 6±0.9 and 7.2±4.2 percent. The results fromthis study show compounds IV, V, X, and XI have surprisingly much lowerintravenous plasma clearance than dasatinib and compound III. This datais consistent with the in vitro metabolic stability data which showedcompounds IV, V, X, and XI to be significantly more stable than eitherdasatinib or compound III, and with significantly lower intrinsicclearance values.

Additionally, compounds IV, V, X, and XI were found to have asurprisingly significantly lower intravenous volume of distributionvalues than dasatinib and compound III, suggesting these compounds arenot as widely distributed to tissues as compared to dasatinib andcompound III. Further, this suggests these compounds have lowerpotential than dasatinib for drug-induced organ toxicity.

These results indicate that chemical substitution on the pyrimidin-4-ylgroup to produce compounds IV, V, X, and XI results in unexpected andsignificant changes in the pharmacokinetic profile of these novelcompounds as compared to dasatinib.

In Vivo Tissue Distribution: A study was conducted in mice to determinethe ratio of parent compound concentrations in lung tissue versus plasmafor compounds X, XI, and dasatinib dosed by oral gavage (see Example 19for experimental conditions). Results are shown in FIGS. 4 and 5.

Compounds X and XI unexpectedly showed significantly lower rats forparent compound concentration levels in lung tissue compared to theirplasma concentrations as compared to dasatinib at all time points tested(FIGS. 4 and 5). These results show that compounds X and XI distributeless than dasatinib into lung tissue.

Dasatinib is associated with severe respiratory toxicity such as pleuraleffusion and pulmonary hypertension, which have been attributed to theaccumulation of dasatinib in lung tissue, (Quintás-Cardama A et al.Pleural effusion in patients with chronic myelogenous leukemia treatedwith dasatinib after imatinib failure. J Clin Oncol 2007;25(25):3908-14; Wang X et al. Differential effects of dosing regimen onthe safety and efficacy of dasatinib: retrospective exposure-responseanalysis of a Phase III study. Clin Pharmacol, 2013; 5; 85-97;Guignabert C et al. Dasatinib induces lung vascular toxicity andpredisposes to pulmonary hypertension. J Clin Invest 2016; 126(9)207-18; Iurlo A, et al. Pleural effusion and molecular response indasatinib-treated chronic myeloid leukemia patients in a real-lifeItalian multicenter series. Ann Hematol. 2017 Oct. 2. Doi;10.1007/soo277-017-3144-1).

Therefore, the unexpected significantly lower distribution of compoundsX and XI into lung tissue from plasma is suggestive of lower potentialof these compounds to accumulate in lung tissue, and therefore, a lowerpotential for drug-induced lung toxicity as compared to dasatinib.

Combination Therapy

In one aspect, the composition to be administered can include aplurality of different pharmacologically active compounds which caninclude a plurality of compounds of the invention including one or morecompounds of formulas I, II, III, IV, and/or V, preferably one or moreof compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII,and/or XIV (compounds I-XIV), and more preferably one or more ofcompounds IV, V, X, and/or XI, including salts, prodrugs, and/or isomersthereof, and other therapeutically effective agents for the same diseaseor condition, wherein the compounds have an additive or a synergisticeffect on the disease indication.

In one preferred aspect, the invention provides methods for treating akinase dysfunction-mediated disease or condition in an animal or humansubject, wherein the method involves administering to the subject aneffective amount of one or more compounds having formulas I, II, III,IV, and/or V, preferably one or more of compounds I, II, III, IV, V, VI,VII, VIII, IX, X, XI, XII, XIII, and/or XIV (compounds I-XIV), and morepreferably one or more of compounds IV, V, X, and/or XI, salts, prodrugsand/or isomers thereof, in combination with one or more other therapiesfor treating the same disease or condition. Other therapies may includemedical procedures (such as surgeries), therapeutic agents, and/orradiation. Therapeutic agents include chemotherapeutic agents,biologics, and immunotherapeutics. Combination therapy can includeadministration of one or more of compounds described herein with one ormore other therapeutics at different times or simultaneousadministration. In some embodiments, dosages may be modified for one ormore of the compounds of the invention or other therapeutics used incombination, such modifications being a reduction in the dose amountsrelative to a compound or therapy used alone.

It is understood that use in combination includes use with other medicalprocedures, therapeutics, and therapies where the other therapy or drugmay be administered at different times, within a short time period, suchas within 2, 3, or 4-24 hours, or within a longer time period, such a1-2 days, 2-4 days, 4-7 days, or 1-4 weeks. Use of the compounds of theinvention can be in combination with a medical procedure such assurgery, performed on the subject once or infrequently, where thecompounds are administered within a short time or longer time before orafter the medical procedure.

Administration

The methods and compounds will typically be used in therapy for humansubjects with a kinase-mediated disease or condition. However, they mayalso be used to treat similar or identical indications in other animalsubjects. In this context, the terms “subject” and “animal subject” andthe like refer to human and non-human vertebrates, i.e., mammals, suchas non-human primates, sports and commercial animals, e.g., equines,bovines, porcine, ovines, rodents, and pets, e.g., canines and felines.

Compounds of formulas I, II, III, IV, and V, preferably compounds I, II,III, IV, V, VI, VII, VIII, IX, X, XI XII, XIII, and/or XIV, and morepreferably compounds IV, V, X, and XI may in some cases form salts whichare also within the scope of this invention. The term “salts(s)”, asemployed herein, denotes acidic and/or basic salts formed with inorganicand/or organic acids and bases.

Exemplary acid addition salts include acetates (such as those formedwith acetic acid or trihaloacetic acid, for example trifluoroaceticacid), adipates, alginates, ascorbates, aspartates, benzoates, citrates,camphorates, camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates,hydrochlorides, hydrobromides, 2-hydroxyethanesulfanates, lactates,maleates, methanesulfonates, nicotinates nitrates, oxalates, pectinates,phosphates, picrates, salicylates, propionates, tartrates, thiocyantes,toluenesulfonates, and the like.

Exemplary basic salts include ammonium salts alkali metal salts suchsodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, and salts with organic bases (for exampleorganic amines), and the like.

Compounds of formulas I, II, III, IV, and V, including compounds I, II,III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and/or XIV (compoundsI-XIV), salts, prodrugs, and/or isomers thereof, can be administeredintravenously, intramuscularly, subcutaneously, orally, transdermally,transmucosally, rectally, or by inhalation. In the case of intravenousadministration, the dose may be administered as a bolus or infusion.

Pharmaceutical compositions for oral use can be obtained, for example,by combining one or more compounds of formula I, II, III, IV, and/or V,preferably one or more of compounds I, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, XIII, and/or XIV, and more preferably one or more ofcompounds IV, V, X, and/or XI, salts, prodrugs, and/or isomers thereof,with solid excipients, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipients are,in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations, for example, maizestarch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose (CMC), and/or polyvinylpyrrolidone (PVP:povidone). If desired, disintegrating agents may be added, such as thecross-linked polyvinylpyrrolidone, agar, or alginic acid, or a saltthereof such as sodium alginate.

For injection, the compounds of formula I, II, III, IV, and V,preferably compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,XIII and XIV, and more preferably compounds IV, V, X, and XI, salts,prodrugs, and/or isomers thereof, are formulated in sterile liquidsolutions, preferably in physically compatible buffers or solutions,such as saline solution Hank's solution, or Ringer's solution. Inaddition, the compounds may be formulated in solid form and re-dissolvedor suspended immediately prior to use. Lyophilized forms can also beproduced.

The administration of the compounds described herein can occursimultaneously or sequentially with chemotherapy or radiation. It isunderstood that administration of other therapeutics or drugs to treat amedical disease or condition can be by a different route ofadministration or by the same route of administration.

In another aspect, the use in combination therapy for any route ofadministration includes delivery of compounds of the invention and oneor more other drug therapeutics delivered by the same route ofadministration together in any formulation, or administered together,within 1 hour, 2 hours, 3 hours, up to 24 hours, in separateformulations, or by different routes of administration.

The invention also provides for a pharmaceutical combination, e.g., akit, comprising (a) a first agent which is a compound of the inventionas disclosed herein, in free form or in pharmaceutically acceptable saltform, and (b) at least one co-agent. The kit can include instructionsfor administration.

General Synthetic Methods

The present invention also includes processes for the preparation of thenovel deuterium-enriched and non-enriched compounds of the invention. Inthe reactions described, it can be necessary to protect reactivefunctional groups, for example hydroxy, amino, imino, thio, or carboxygroups, where these are desired in the final product, to avoid theirunwanted participation in the reactions. Conventional protecting groupscan be used in accordance with standard practice, for example, see T WGreene and P G M Wutsin Protective Groups in Organic Chemistry, JohnWiley and Sons, 1991.

Compounds of formulas I, II, III, IV, and V, including the exemplarycompounds, can generally be synthesized by making appropriatemodifications to reagents of scheme 1 (and other applicable schemes)below as would be understood by the person of ordinary skill in the art.It is noted that non-deuterated intermediates are generally commerciallyavailable and so the synthesis could be started, for example, atcompound 7 (see e.g. compound 7H in schemes 8-14) using the appropriatecommercially available intermediate.

Compound I can be synthesized by the method show Scheme 1.

The initial step of the chemical process involves reacting4-bromo-2-chloro-6-methylaniline (1) with allyl bromide to formintermediate 2. To intermediate 2 in dry tetrahydrofuran under nitrogengas at about −70° C. is added dropwise n-butyl (about 1.5 moles n-butyllithium to 1 mole intermediate 2). After about 40 minutes theintermediate lithium complex is quenched with d₁-methanol (CH₃OD; 99%deuterium, #550574; Lot # MKBW0355V, Aldrich, St Louis, Mo.) toselectively incorporate deuterium at the 4 position and giveintermediate 3. The allyl protecting groups are removed by standardprocedure to give the aniline intermediate 4. Intermediate 4 is reactedwith 3-ethoxyacryloyl chloride to form the acrylamide intermediate 5which is then treated with N-bromosuccinimide and thiourea to form thethiazole intermediate 6. The thiazole intermediate 6 is treated with thebase sodium hydride followed by addition of4,6-dichloro-2-methylpyrimidine to form the2-[(6-chloro-2-methylpyridin-4-yl)amino]-N-(2-chloro-4-deutero-6-methylphenyl)thiazole-5-carboxamide(intermediate 7). Intermediate 7 is reacted with piperidine andN,N-diisopropylethylamine to form the desired product compound I (8).

Compound II can be synthesized by the method shown in Scheme 2.

In the synthesis of compound II, the first 6 steps in the method areidentical to those used in the synthesis of compound I to produceintermediate 7. The last step in the synthesis uses 4-hydroxypiperidineand N,N-diisopropylethylamine to react with intermediate 7 to form thedesired product compound II.

Compound III can be synthesized by the method shown in Scheme 3.

In the synthesis of compound III, the first 6 steps in the method areidentical to those used in the synthesis of compound I to produceintermediate 7. The last step in the synthesis uses1-(2-hydroxyethyl)piperazine (Sigma-Aldrich; St Louis, Mo.) andN,N-diisopropylethylamine to react with intermediate 7 to form thedesired addition product compound III.

Compound IV can be synthesized by the method shown in Scheme 4.

In the synthesis of compound IV, the first 6 steps in the method areidentical to those used in the synthesis of compound I to produceintermediate 7. Intermediate 7 is react with (S)-3-hydroxypyrrolidineand N,N-diisopropylethylamine to form the desired product compound IV.

Compound V can be synthesized by the method shown in Scheme 5.

In the synthesis of compound V, the first 6 steps in the method areidentical to those used in the synthesis of compound I to produceintermediate 7. Intermediate 7 is reacted with (R)-3-hydroxypyrrolidineand N,N-diisopropylethylamine to form the desired product compound V.

Compound VI can be synthesized by the method shown in Scheme 6.

In the synthesis of compound VI, the first 6 steps in the method areidentical to those used in the synthesis of compound I to produceintermediate 7. Intermediate 7 is reacted with tert-butyl(S)-pyrrolidin-3-ylcarbamate and N,N-diisopropylethylamine in dioxane toform compound 300-98. Compound 300-98 is deprotected to produce theproduct compound VI.

Compound VI can be synthesized by the method shown in Scheme 7.

In the synthesis of compound VII, the first 6 steps in the method areidentical to those used in the synthesis of compound I to produceintermediate 7. Intermediate 7 is reacted with tert-butyl(R)-pyrrolidin-3-ylcarbamate and N,N-diisopropylethylamine in dioxane toform compound 300-100. Compound 300-100 is deprotected to produce theproduct compound VII.

Compound VIII can be synthesized by the method shown in Scheme 8.

In the synthesis of compound VIII, commercially available intermediate7H (Combi-Blocks, Inc., San Diego, Calif.) is reacted with piperidineand N,N-diisopropylethylamine in dioxane to form the desired productcompound VIII.

Compound IX can be synthesized by the method shown in Scheme 9.

In the synthesis of compound IX, commercially available intermediate 7His reacted with 4-hydroxypiperidine and N,N-diisopropylethylamine indioxane to form the desired product compound IX.

Compound X can be synthesized by the method shown in Scheme 10.

In the synthesis of compound X, commercially available intermediate 7His reacted with (S)-pyrrolidin-3-ol hydrochloride andN,N-diisopropylethylamine in dioxane to form the desired productcompound X.

Compound XI can be synthesized by the method shown in Scheme 11.

In the synthesis of compound X, commercially available intermediate 7His reacted with (R)-pyrrolidin-3-olhydrochloride andN,N-diisopropylethylamine in dioxane to form the desired productcompound XI.

Compound XI can be synthesized by the method shown in Scheme 12.

In the synthesis of compound XII, commercially available intermediate 7His reacted with tert-butyl (S)-pyrrolidin-3-ylcarbamate andN,N-diisopropylethylamine in dioxane to form intermediate 300-92.Intermediate 300-92 is deprotected to produce compound XII.

Compound XIII can be synthesized by the method shown in Scheme 13.

In the synthesis of compound XIII, commercially available intermediate7H is reacted with tert-butyl (R)-pyrrolidin-3-ylcarbamate andN,N-diisopropylethylamine in dioxane to form intermediate 300-92.Intermediate 300-92 is deprotected to produce compound XII.

Compound XIV can be synthesized by the method shown in Scheme 14.

In the synthesis of compound XIV, commercially available intermediate 7His reacted with pyrrolidine and N,N-diisopropylethylamine in dimethylsulfoxide (DMSO) to form compound XIV.

EXAMPLES

Examples related to the present invention are described below. In morecases, alternative techniques can be used. The examples are intended tobe illustrative and are not limiting or restrictive to the scope of theinvention. In some examples, the mass spectrometry results indicatedthat a compound may have more than one value due to the isotopedistribution of an atom in the molecule, such as a compound having abromo or chloro substituent.

Example 1 Synthesis ofN-(2-chloro-4-deuterio-6-methyl-phenyl)-2-[[2-methyl-6-(1-piperidyl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound I)

Preparation of N,N-diallyl-4-bromo-2-chloro-6-methylaniline (2): To a250 mL flask were added 4-bromo-2-chloro-6-methylaniline (3 g, 13.61mmol), dimethyl formamide (DMF) (50 mL) and sodium carbonate (6.37 g,60.09 mmol, 4.4 eq) at 0° C. With stirring, allyl bromide (9.4 mL,108.62 mmol, 8 eq) was added dropwise at 0-5° C. under nitrogen. Afteraddition, the mixture was stirred at room temperature for 20 min andheated at 120° C. under nitrogen for 3 h when TLC analysis showed nopresence of the starting material. The mixture was then cooled to roomtemperature and poured into cold water followed by extraction with ethylacetate (EtOAc) (2×150 mL). The combined organic layers were washed withbrine (3×100 mL), dried (sodium sulfate, Na₂SO₄), and concentrated underreduced pressure to get a black-brown liquid residue, which was purifiedby column chromatography (hexanes only) to give compound 2 (3.9 g, 95%)his a pale brown liquid.

Preparation of N,N-diallyl-2-chloro-4-deutero-6-methylaniline (3): To asolution of compound 2 (2 g 6.65 mmol) in tetrahydrofuran (THF) (40 mL)at −70° C. under nitrogen was added 2.5 M solution of n-butyl lithium (4ml, 10 mmol) dropwise. After addition, the mixture was stirred at −70°C. for 40 min and then quenched by addition of deuterated methanol(MeOD) (2 mL, 49.2 mmol, 99% deuterium, #550574; Lot # MKBW0355V,Aldrich, St Louis, Mo.) dropwise. The reaction mixture was then stirredfrom −70° C. to −20° C. over 40 min when thin layer chromatograph (TLC)analysis (hexanes only) showed the reaction was complete. The mixturewas poured into cold water (100 mL) followed by extraction with EtOAc(2×100 mL). The combined organic layers were washed with brine, dried(Na₂SO₄), and concentrated under reduced pressure. The residue waspurified by column chromatography (hexanes only, then EtOAc:hexanes;1:20) to give compound 3 (1.38 g, 93%) as a pale yellow liquid. ¹HNuclear magnetic resonance (NMR) (CDCl3): 7.20 (s, 1H), 7.09 (s, 1H),5.95 (m, 2H), 5.21-5.20 (m, 4H), 3.79 (d, 4H), 2.41 (s, 3H). ¹H NMRshowed absence of the proton signal at the 4 position of compound 3.

Preparation of 2-chloro-4-deutero-6-methylaniline (4): To a stirringsolution of the aniline 3 (3 g, 13.31 mmol) in DCM (130 mL) were addedN,N.dimethylbarbituric acid (8.3 g, 53.16 mmol, 4 eq) and Pd(PPh₃)₄ (0.5g 0.43 mmol, 0.032 eq). The mixture was heated at reflux under nitrogenfor 4 h. TLC analysis (EtOAc:hexanes; 1:9) showed the reaction wascomplete. After the mixture was cooled to room temperature andconcentrated under reduced pressure. The residue was dissolved in EtOAc(120 mL) and the organic layer was washed with 10 sodium bicarbonate(NaHCO₃) solution (4×60 mL) dried (Na₂SO₄), and concentrated. The crudeproduct was purified by column chromatography (EtOAc:hexanes; 1:9) togive the desired aniline 4 (1.67 g, 88%) as a pale brown liquid.

Preparation of N-(2-chloro-4-deutero-6-methylphenyl)-3-ethoxyacrylamide(5): To a mixture of compound 4 (460 mg, 3.23 mmol), pyridine (0.4 mL,4.95 mmol, 1.5 eq), and THF (25 mL) at 0-5° C. was added3-ethoxyacryloyl chloride (666 mg, 4.95 mmol, 1.5 eq) dropwise. Afteraddition, the mixture was stirred at room temperature under nitrogenovernight. TLC analysis (EtOAc:hexanes; 1:2) showed absence of startingmaterial. EtOAc (80 mL) and water (80 mL) were added to the mixture andthe organic layer separated and washed with 1N hydrochloric add (HCl)solution, water, and 5% NaHCO₃ solution, dried (Na₂SO₄), andconcentrated to give the compound 6 as a white solid, which was used inthe next step without purification.

Preparation of2-amino-N-(2-chloro-4-deutero-6-methylphenyl)thiazole-5-carboxamide (6):To a mixture of the acrylamide 5 (900 mg, 3.74 mmol), 1,4-dioxane (7mL), and water (7 mL) was added N-bromosuccinimide (730 mg, 4.10 mmol1.1 eq) at 0° C. The slurry was stirred at room temperature for 3 h.Thiourea (285 mg, 3.75 mmol, 1 eq) was added and the mixture heated to80° C. After 3 h, the mixture was cooled to room temperature followed byaddition of concentrated ammonium hydroxide solution (1 mL). Afterstirring, EtOAc (80 mL) and water (80 mL) were added to the mixture. Theorganic layer was separated and the aqueous layer was extracted withEtOAc (80 mL). The combined organic layers were washed with water, dried(Na₂SO₄), and concentrated. The residue was subjected to columnchromatography (EtOAc-hexanes, 1:4 to 1:2) to give compound 6 (0.82 g,82%) as a pale brown solid. Liquid chromatography-mass spectrometry(LC-MS) analysis showed a protonated parent ion at 269.14 (M+H).

Preparation of2-[(6-chloro-methylpyrimidin-4-yl)amino]-N-(2-chloro-4-deutero-6-methylphenyl)thiazole-5-carboxamide(7): To a mixture of sodium hydride (60%, 67 mg, 1.67 mmol) and THF (15mL) at 0° C. was added compound 6 (150 mg, 0.56 mmol) in portions. Themixture was stirred at 0° C. for 30 min and a solution of4,8-dichloro-2-methylpyrimidine (109 mg 0.67 mmol, 1.2 eq) was addeddropwise. The resulting mixture was stirred at room temperature for 3 hwhen LC-MS analysis showing absence of starting material. The mixturewas concentrated to provide the crude compound 7, which was used in nextstep without purification.

Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-(piperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(8, compound 1): To a mixture of the crude compound 7 (about 0.56 mmol)in dioxane (10 mL) was added piperidine (143 mg, 1.68 mmol, 3 eq) andN,N-diisopropylethylamine (DIEA) (217 mg, 1.68 mmol, 3 eq) at roomtemperature. The mixture was stirred at 85-90° C. under nitrogen for 20h. LC-MS analysis showed the product peak. The mixture was not a clearsolution. The mixture was concentrated to dryness and suspended in 50 mLacetonitrile containing 20% HPLC grade water. The mixture was thencentrifuged at 4000 rpm for 15 minutes. The pellet was re-suspended inacetonitrile and centrifuged again at 4000 rpm for 15 minutes. The finalpellet was dried under nitrogen, and recovered as an off-white solid ofthe target Compound I (compound 8) (12 mg). The purity of the finalproduct was determined to be greater than 95% by liquidchromatograph-ultraviolet-mass spectrometry (LC-UV-MS). LC-MS: 444.14(M+H); ¹H NMR (DMSO-d₆): 11.52 (s, 1H, NH), 9.82 (s, 1H, NH), 8.18 (s,1H) 7.39 (s, 1H), 7.21 (s, 1H), 6.00 (s, 1H), 3.55 (m, 4H), 3.22 (s,3H), 2.43 (s, 3H), 1.65 (m, 6H). ¹H NMR showed absence of a protonsignal at the 4-position of compound I.

Example 2 Preparation ofN-(2-chloro-4-deuterio-6-methyl-phenyl)-2-[[6-(4-hydroxy-1-piperidyl)-2-methyl-pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound 11)

Compound II was synthesized starting from2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-4-deutero-6-methylphenyl)thiazole-5-carboxamide(7) by the synthetic procedure shown in Scheme 2.

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-4-deutero-6methylphenyl)thiazole-5-carboxamide(7) was prepared in six steps starting from4-bromo-2-chloro-6-methylaniline as described in Example 1.

Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-(4-hydroxypiperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(compound II): To a mixture of the crude compound 7 (about 0.56 mmol) indioxane (10 mL) was added 4-hydroxypiperidine (170 mg, 1.68 mmol, 3 eq)and DIEA (217 mg, 1.68 mmol, 3 eq) at room temperature. The mixture wasstirred at 85-90° C. under nitrogen for 20 h. LC-MS analysis showed theproduct peak. The mixture was not a clear solution. The mixture wasconcentrated to dryness followed by suspending in 50 mL acetonitrile.The mixture was centrifuged at 4000 rpm for 15 min. The pellet wasdissolved in methanol, and purified with column chromatography(methanol-methylene chloride, 1:9). The column chromatograph fractionscontaining only compound II were combined and dried under a nitrogenflow and recovered as an off-white solid of the target compound II (17mg). The purity of the final product was determined to be greater than95% by LC-UV-MS. LC-MS: 460.14 (M+H); ¹H NMR (DMSO-d₆): 11.42 (s, 1H,NH), 9.83 (s, 1H, NH), 8.19 (s, 1H), 7.40 (s, 1H), 7.24 (s, 1H), 6.05(s, 1H), 4.78 (s, 1H), 3.95 (m, 2H), 3.75 (m, 1H), 3.22 (s, 3H), 3.18(m, 2H), 2.43 (s, 3H), 1.85 (m 2H), 1.35 (m, 2H). ¹H NMR showed absenceof a proton signal at the 4-position of compound II.

Example 3 Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-(4-(2-hydroxyethyl)piperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound III)

To a mixture of the crude compound 7 (about 0.56 mmol) and dioxane (10mL) were added 4-(2-hydroxyethyl)piperidine (219 mg, 1.68 mmol, 3 eq)and DIEA (217 mg, 1.68 mmol, 3 eq) at room temperature. The mixture wasthen stirred at 85-90° C. under nitrogen for 20 h. LC-MS analysis showedthe product peak. The mixture was not a clear solution. The mixture wascooled to room temperature, concentrated under reduced pressure todryness, and suspended in 50 mL acetonitrile containing 20% HPLC gradewater. The mixture was then centrifuged at 4000 rpm for 15 min. Thepellet was re-suspended in acetonitrile and centrifuged again at 4000rpm for 15 min. The final pellet was dried under nitrogen, and recoveredas an off-white solid of the target compound III (about 40 mg) as anoff-white solid. LC-MS: 489.16 (M+H); ¹H NMR (DMSO-d₆): 11.42 (s, 1H,NH), 9.83 (s, 1H, NH), 8.20 (s, 1H), 7.40 (s, 1H), 7.24 (s, 1H), 6.05(s, 1H), 4.42 (m, 1H), 3.35 (m, 2H), 2.48 (m, 8H), 2.40 (s, 3H), 2.15(m, 2H).

Example 4 Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-((S)-3-hydroxypyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound IV)

To a mixture of the crude compound 7 (about 0.56 mmol) and dioxane (10mL) were added (S)-pyrrolidin-3-ol hydrochloride (208 mg, 1.68 mmol, 3eq) and DIEA (400 mg, 3.1 mmol, 5.5 eq) at room temperature. The mixturewas then stirred at 85-90° C. under nitrogen for 6 h. LC-MS analysisshowed the product peak. The mixture was not a clear solution. Themixture was cooled to room temperature and concentrated to dryness underreduced pressure, and the resultant residue was suspended in 50 mLacetonitrile, and centrifuged at 4000 rpm for 15 min. The pellet wasthen suspended in cooled 80% acetonitrile, and centrifuged at 4000 rpmfor 15 min. The pellet was re-suspended in cooled 80% acetonitrile, andcentrifuged at 4000 rpm for 15 min. The supernatants were combined andconcentrated to dryness to afford the target compound IV (about 60 mg)as an off-white solid. LC-MS: 446 (M+H); ¹H NMR (DMSO-d₆): 11.40 (s, 1H,NH), 9.83 (s, 1H, NH), 8.19 (s, 1H), 7.40 (s, 1H), 7.24 (s, 1H), 5.80(s, 1H), 4.98 (s, 1H), 4.35 (s, 1H), 2.53 (s, 3H), 2.20 (s, 2H), 2.12(s, 2H), 1.85 (m, 2H).

Example 5 Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-((R)-3-hydroxypyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound V)

To a mixture of the crude compound 7 (about 0.56 mmol) and dioxane (10mL) were added (R)-pyrrolidin-3-ol hydrochloride (208 mg, 1.68 mmol, 3eq) and DIEA (400 mg, 3.1 mmol, 5.5 eq) at room temperature. The mixturewas then stirred at 85-90° C. under nitrogen for 6 h. LC-MS analysisshowed the product peak. The mixture as not a clear solution. Themixture was cooled to room temperature and concentrated to dryness underreduced pressure, and the resultant residue was suspended in 50 mLacetonitrile, and centrifuged at 4000 rpm for 15 min. The pellet wasthen suspended in cooled 80% acetonitrile, and centrifuged at 4000 rpmfor 15 min. The pellet was re-suspended in cooled 80% acetonitrile, andcentrifuged at 4000 rpm for 15 min. The supernatants were combined andconcentrated to dryness to afford the target compound V (about 103 mg)as an off-white solid. LC-MS: 446 (M+H); ¹H NMR (DMSO-d₆): 11.40 (s, 1H,NH), 9.83 (s, 1H, NH), 8.19 (s, 1H), 7.40 (a, 1H), 7.24 (s, 1H), 5.80(s, 1H), 4.98 (s, 1H), 4.35 (s, 1H), 2.53 (s, 3H), 2.20 (s, 2H), 2.12(s, 2H), 1.85 (m, 2H).

Example 6 Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-((S)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound VI, 300-101)

Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-((S)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound VII, 300-101). To a mixture of 300-97 (200 mg, 0.51 mmol) anddioxane (8 mL) were added tert-butyl (S)-pyrrolidin-3-ylcarbamate (186mg, 1 mmol, 2 eq) and DIEA (147 mg, 1.14 mmol, 2 eq) at roomtemperature. The mixture was then stirred at 90-91° C. under nitrogenfor 12 h. LC-MS analysis showed the product peak. The mixture was cooledto room temperature and concentrated under reduced pressure afteraddition of methanol (4 mL), the mixture was re-concentrated to give theintermediate 300-100 as a grey solid. LC-MS: 545.12 (M+H).

To the crude sample (300-100) was added DCM (4 mL). The mixture wascooled to 5° C., and then a mixture of TFA-DCM (1:1, 5 mL) was addeddropwise. After addition, the mixture was stirred at room temperaturefor 3 h and concentrated under reduced pressure. The residue was mixedwith methanol (4 mL), followed by addition of triethylamine (2 mL) andstirring a while. The mixture was concentrated to dryness, thensuspended in 50 mL distilled water, and centrifuged at 4000 rpm for 15min. The pellet was re-suspended with 50 mL distilled water, and thencentrifuged at 4000 rpm for 15 min. The pellet was further suspendedwith 100 mL 80% acetonitrile, and centrifuged at 4000 rpm for 15 min.The supernatant was evaporated to dryness to afford the target compoundVI (300-101) (approximately 108 mg) as an off-white solid. LC-MS: 445(M+H); ¹H NMR (DMSO-d₆): 9.83 (s, 1H, NH), 8.19 (s, 1H), 7.30 (s, 1H),7.19 (s, 1H), 5.80 (s, 1H), 3.60-3.35 (m, 2H), 2.43 (s, 3H), 2.20 (s,2H), 2.12 (s, 2H), 1.85 (m, 2H).

Example 7 Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-((R)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound VII, 300-99)

Preparation of2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-4-deutero-6-methylphenyl)thiazole-5-carboxamide(300-97). To a stirred mixture of the starting material 300-77-2 (200mg, 0.74 mmol), 4,6-dichloro-2-methylpyrimidine (147 mg, 0.90 mmol, 1.2eq), and THF (4 mL) was added a solution of sodium tertiary-butoxide inTHF (2M, 1.31 mL, 2.62 mmol, 3.5 eq) dropwise at 0-5° C. After addition,the mixture stirred at room temperature for 1.5 h and re-cooled to 0-5°C. 2N HCl solution (1 mL) was added dropwise. The mixture was stirredfor 15 min and concentrated under reduced pressure. The residue wasmixed with EtOAc-hexane (1:1) and stirred for 5 min. The solid wasfiltered and washed with EtOAc-hexane (1:1) and dried to give a yellowsolid (210 mg), which was used in the next step without purification.LC-MS: 395.03 (M+H).

Preparation ofN-(2-chloro-4-deutero-6-methylphenyl)-2-[[2-methyl-6-((R)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound VI, 300-99). To a mixture of 300-97 (200 mg, 0.51 mmol) anddioxane (8 mL) were added tert-butyl (R)-pyrrolidin-3-ylcarbamate (186mg, 1 mmol, 2 eq) and DIEA (147 mg, 1.14 mmol, 2 eq) at roomtemperature. The mixture was then stirred at 90-91° C. under nitrogenfor 12 h. LC-MS analysis showed the product peak. The mixture was cooledto room temperature and concentrated under reduced pressure. Afteraddition of methanol (4 mL), the mixture was re-concentrated to give theintermediate 300-98 as a grey solid. LC-MS: 545.12 (M+H).

To the crude sample (300-98) was added dichloromethane (DCM) (4 mL). Themixture was cooled to 5° C., and then a mixture of trifluoroacetic acid(TFA)-DCM (1:1, 5 mL) was added dropwise. After addition, the mixturewas stirred at room temperature for 3 h and concentrated under reducedpressure. The residue was mixed with methanol (4 mL), followed byaddition of triethylamine (2 mL) and stirred. The mixture wasconcentrated to dryness, then suspended in 50 mL distilled water, andcentrifuged at 4000 rpm for 15 min. The pellet was re-suspended with 50mL distilled water, and then centrifuged at 4000 rpm for 15 min. Thepellet was further suspended with 100 mL 80% acetonitrile, andcentrifuged at 4000 rpm for 15 min. The supernatant was evaporated todryness to afford the target compound VII (300-99) as an off-white solid(˜105 mg). LC-MS: 445 (M+H); ¹H NMR (DMSO-d₆): 9.83 (s, 1H, NH), 8.19(s, 1H), 7.30 (s, 1H), 7.19 (s, 1H), 5.80 (s, 1H), 3.60-3.35 (m, 2H)2.43 (s, 3H) 2.20 (s, 2H), 2.12 (s, 2H), 1.85 (m, 2H).

Example 8 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-(piperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound VIII, H-20)

Preparation ofN-(2-chloro-8-methylphenyl)-2-[[2-methyl-6-(piperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound VIII, H-20). To a mixture of the starting material 7H (150 mg,0.38 mmol), dioxane (8 mL) were added piperidine (97 mg, 1.14 mmol, 3eq) and DIEA (147 mg, 1.14 mmol, 3 eq) at room temperature. The mixturewas stirred at 90-91° C. under nitrogen for 15 h. LC-MS analysis showedthe product peak. The mixture was not a clear solution. The mixture wasconcentrated to dryness and suspended in 50 mL acetonitrile containing20% HPLC grade water. The mixture was then centrifuged at 4000 rpm for15 min. The pellet was re-suspended in acetonitrile and centrifugedagain at 4000 rpm for 15 min. The final pellet was dried under nitrogento afford the target compound VIII (H-20) (˜129 mg) as an off-whitesolid. LC-MS: 443.14 (M+H); ¹H NMR (DMSO-d₆): 11.52 (s, 1H, NH), 9.82(s, 1H, NH), 8.18 (s, 1H), 7.39 (m 1H), 7.21 (m, 2H), 6.00 (s, 1H), 3.55(m, 4H), 3.22 (s, 3H), 2.43 (s, 3H), 1.65 (m, 6H).

Example 9 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-(4-hydroxypiperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound IX, H-21)

Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-(4-hydroxypiperidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(H-21). To a mixture of the starting material 7H (150 mg, 0.38 mmol) anddioxane (8 mL) were added 4-hydroxypiperidine (115 mg, 1.14 mmol, 3 eq)and DIEA (147 mg, 1.14 mmol, 3 eq) at room temperature. The mixture wasthen stirred at 90-92° C. under nitrogen for 10 h. LC-MS analysis showedthe product peak. The mixture was not a clear solution. The mixture wasconcentrated to dryness and suspended in 50 mL acetonitrile containing20% HPLC grade water. The mixture was then centrifuged at 4000 rpm for15 min. The pellet was re-suspended in acetonitrile and centrifugedagain at 4000 rpm for 15 min. The final pellet was dried under nitrogento afford the target compound IX (H-21) (130 mg) as an off-white solid.LC-MS: 459.14 (M+H); ¹H NMR (DMSO-d₆): 11.42 (s, 1H, NH), 9.83 (s, 1H,NH), 8.19 (s, 1H), 7.40 (m, 1H), 7.24 (m, 2H), 6.05 (s, 1H) (s, 1H),3.95 (m, 2H), 3.75 (m, 1H), 3.22 (s, 3H), 3.18 (m, 2H), 2.43 (s, 3H),1.85 (m, 2H), 1.35 (m, 2H).

Example 10 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((S)-3-hydroxypyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound X, H-31, 300-89)

Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((S)-3-hydroxypyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound XI, H-31, 300-89). To a mixture of the starting material 7H(150 mg, 0.38 mmol) and dioxane (8 mL) were added (S)-pyrrolidin-3-olhydrochloride (141 mg, 1.14 mmol, 3 eq) and DIEA (245 mg, 1.90 mmol, 5eq) at room temperature. The mixture was then stirred at 90-92° C. undernitrogen for 12 h. LC-MS analysis showed the product peak. The mixturewas not a clear solution. The mixture was cooled to room temperature andconcentrated to dryness under reduced pressure, and the resultantresidue was suspended in 50 mL acetonitrile, and centrifuged at 4000 rpmfor 15 min. The pellet was then suspended in cooled 80% acetonitrile,and centrifuged at 4000 rpm for 15 min. The pellet was re-suspended incooled 80% acetonitrile, and centrifuged at 4000 rpm for 15 min. Thesupernatants were combined and concentrated to dryness to afford thetarget compound X (H-31) (105 mg) as an off-white solid. LC-MS: 445(M+H); ¹H NMR (DMSO-d₆): 11.40 (s, 1H, NH), 9.83 (s, 1H, NH), 8.19 (s,1H), 7.40 (m, 1H), 7.24 (m, 2H), 5.80 (s, 1H), 4.98 (s, 1H), 4.35 (s,1H), 2.53 (s, 3H), 2.20 (s, 2H), 2.12 (s, 2H), 1.85 (m, 2H).

Example 11 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((R)-3-hydroxypyrrolidin-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound XI, H-30, 300-87)

Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((R)-3-hydroxypyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound X, H-30, 300-87). To a mixture of the starting material 7H(150 mg, 0.38 mmol) and dioxane (8 mL) were added (R)-pyrrolidin-3-ol(100 mg, 1.1 mmol, 3 eq) and DIEA (147 mg, 1.14 mmol, 3 eq) at roomtemperature. The mixture was then stirred at 90-92° C. under nitrogenfor 12 h. LC-MS analysis showed the product peak. The mixture was not aclear solution. The mixture was cooled to room temperature andconcentrated to dryness under reduced pressure, and the resultantresidue was suspended in 50 mL acetonitrile, and centrifuged at 4000 rpmfor 15 min. The pellet was then suspended in cooled 80% acetonitrile,and centrifuged at 4000 rpm for 15 min. The pellet was re-suspended incooled 80% acetonitrile, and centrifuged at 4000 rpm for 15 min. Thesupernatants were combined and concentrated to dryness to afford thetarget compound XI (H-30) (˜75 mg) a an off-white solid. LC-MS: 445(M+H); ¹H NMR (DMSO-d₆): 11.40 (s, 1H, NH), 9.83 (s, 1H, NH), 8.19 (s,1H), 7.40 (m, 1H), 7.24 (m, 2H), 5.80 (s, 1H), 4.98 (s, 1H), 4.35 (s,1H), 2.53 (s, 3H), 2.20 (s, 2H), 2.12 (s, 2H), 1.85 (m, 2H).

Example 12 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((S)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound XII, H-41, 300-93)

Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((S)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(H-41, 300-93). To a mixture of the starting material 7H (150 mg, 0.38mmol) and dioxane (8 mL) were added tert-butyl pyrrolidin-3-ylcarbamate(142 mg, 0.76 mmol, 2 eq) and DIEA (147 mg, 1.14 mmol, 3 eq) at roomtemperature. The mixture was then stirred at 90-91° C. under nitrogenfor 10 h. LC-MS analysis showed the product peak. The mixture was cooledto room temperature and concentrated under reduced pressure. Afteraddition of methanol (4 mL), the mixture was re-concentrated to give theintermediate 300-92 as a grey solid. LC-MS: 544.12 (M+H).

To the crude sample (300-92) was added DCM (4 mL). The mixture wascooled to 5° C., and then a mixture of TFA-DCM (1:1, 5 mL) was addeddropwise. After addition, the mixture was stirred at room temperaturefor 3 h and concentrated under reduced pressure. The residue was mixedwith methanol (4 mL), followed by addition of TEA (2 mL) and stirring awhile. The mixture was concentrated to dryness, then suspended in 50 mLdistilled water, and centrifuged at 4000 rpm for 15 min. The pellet wasre-suspended with 50 mL distilled water, and then centrifuged at 4000rpm for 15 min. The pellet was further suspended with 100 mL 80%acetonitrile, and centrifuged at 4000 rpm for 15 min. The supernatantwas evaporated to dryness to afford the target compound XII (H-41) as anoff-white solid (approximately 88 mg). LC-MS: 444 (M+H); ¹H NMR(DMSO-d₆): 9.83 (s, 1H, NH), 8.19 (s, 1H), 7.40 (m, 1H), 7.24 (m, 2H),5.80 (s, 1H), 3.60-3.35 (m, 2H), 2.53 (s, 3H), 2.20 (s, 2H), 2.12 (s,2H), 1.85 (m, 2H).

Example 13 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((R)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound XIII, H40, 300-91)

Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-((R)-3-aminopyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound XII, H-40, 300-91). To a mixture of the starting material 7H(150 mg, 0.38 mmol) and dioxane (8 mL) were added tert-butyl(R)-pyrrolidin-3-ylcarbamate (142 mg, 0.76 mmol, 2 eq) and DIEA (147 mg,1.14 mmol, 3 eq) at room temperature. The mixture was then stirred at90-91° C. under nitrogen for 12 h. LC-MS analysis showed the productpeak. The mixture was cooled to room temperature and concentrated underreduced pressure. After addition of methanol (4 mL), the mixture wasre-concentrated to give the intermediate 300-90 as a grey solid. LC-MS:544.12 (M+H).

To the crude sample (300-90) was added DCM (4 mL). The mixture wascooled to 5° C., and then a mixture of TFA-DCM (1:1, 5 mL) was addeddropwise. After addition, the mixture was stirred at room temperaturefor 3 h and concentrated under reduced pressure. The residue was mixedwith methanol (4 mL), followed by addition of trimethylamine (TEA) (2mL) and stirring a while. The mixture was concentrated to dryness, thensuspended in 50 mL distilled water, and centrifuged at 4000 rpm for 15min. The pellet, was re-suspended with 50 mL distilled water, and thencentrifuged at 4000 rpm for 15 min. The pellet was further suspendedwith 100 mL 80% acetonitrile, and centrifuged at 4000 rpm for 15 min.The supernatant was evaporated to dryness to afford the target compoundXIII (H-40) as an off-white solid (approximately 96 mg). LC-MS: 444(M+H); ¹H NMR (DMSO-d₆): 9.83 (s, 1H, NH), 8.19 (s, 1H), 7.40 (m, 1H),7.24 (m, 2H), 5.80 (s, 1H), 3.60-3.35 (m, 2H), 2.53 (s, 3H), 2.20 (s,2H), 2.12 (s, 2H), 1.85 (m, 2H).

Example 14 Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-8-(pyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(Compound XIV, H-50)

Preparation ofN-(2-chloro-6-methylphenyl)-2-[[2-methyl-6-(pyrrolidin-1-yl)pyrimidin-4-yl]amino]thiazole-5-carboxamide(H-50): To a mixture of the starting material 7H (300 mg, 0.76 mmol) andDMSO (10 mL) was added pyrrolidine (162 mg, 2.28 mmol, 3 eq) and DIEA(294 mg, 2.28 mmol, 3 eq) and DMAP (3 mg) at room temperature. Themixture was then heated to 110° C. under nitrogen and stirred at 110° C.for 15 h. LC-MS analysis showed the complete reaction. The mixture wasconcentrated to dryness, then suspended in 50 mL distilled water, andcentrifuged at 4000 rpm for 15 min. The pellet was re-suspended with 50mL distilled water, and then centrifuged at 4000 rpm for 15 min. Thepellet was further suspended with 100 mL 80% acetonitrile, andcentrifuged at 4000 rpm for 15 min. The supernatant was evaporated todryness to afford the target compound XIV (H-50) as an off-white solid(approximately 314 mg). LC-MS: 429 (M+H); ¹H NMR (DMSO-d₆): 11.52 (s,1H, NH), 9.82 (s, 1H, NH), 8.18 (s, 1H), 7.39 (m, 1H), 7.21 (m, 2H),5.79 (s, 1H), 3.35 (m, 4H), 2.39 (s, 3H), 2.23 (s, 3H), 1.90 (m, 4H).

Example 15 Protein Kinase Inhibition Studies

Off-chip Mobility Shift Assay (MSA) by Carna Biosciences, Inc (Natick,Mass.) was used for measuring the kinase activity and inhibition.

-   -   1) The 5 μL of ×4 compound solution, 5 μL of ×4        Substrate/ATP/Metal solution, and 10 μL of ×2 kinase solution        were prepared with assay buffer (20 mM HEPES, 0.01% Triton X100,        2 mM DTT, pH 7.5) and mixed and incubated in a well of        polypropylene 384 well microplate for 1 or 5 h (depending on        kinase) at room temperature.    -   2) 70 μL of Termination Buffer (QuickScout Screening Assist MSA;        Carna Biosciences) was added to the well.    -   3) The reaction mixture was applied to a LabChip system (Perkin        Elmer), and the product and substrate peptide peaks were        separated and quantitated.    -   4) The kinase reaction was evaluated by the product ratio        calculated from peak heights of product (P) and substrate (S)        peptides (P/(P+S)).    -   5) The reaction conditions were followed according to assay        protocols of Carna Biosciences Inc (BMA 3F, 1-5-5        Minatojima-Minamimachi Chuo-ku, Kobe 650-0047, Japan;        vww.carnabio.com).    -   6) Data analysis: The readout value of reaction control        (complete reaction mixture) was set as a 0% inhibition, and the        readout value of background (Enzyme (−)) was set as a 100%        inhibition, then the percent inhibition of each test solution        was calculated.

Compound I showed >50% inhibition at 1 nM on the following recombinantkinases: ABL, ABL(E255K), ACK, ARG, BLK, BMX, BTK, DDR2, EPHA1, FGR,FMS, FRK, FYN(isoform a), HCK, LCK, LYNa, PDGFRα, PDGFRβ, SRC, YES.Compound I showed concentration-dependent inhibition of recombinant BTK,ACK and PDGFRα activity with an IC₅₀ of approximately 0.2, 0.5 and 1.4nM respectively. Compound I inhibited recombinant PIK3CA/PIK3R1 activitywith an IC50 of approximately 12 nM.

Compound I showed >50% inhibition at 10 nM on the following recombinantkinases: YES, FRK, SRC, LYNa, FMS, BMX, ABL, FYN(isoform b), FGR, HCK,FYN(isoform a) LCK, DDR2, ARG, ABL(E255K), BTK, EPHA1, BLK, ACK, SRM,PDGFRβ, PIK3CA/PIK3R1 PDGFRα, CSK, KIT(D816V), KIT(D816Y), BRK.

Compound I showed <50% inhibition at 10 nM on the following recombinantkinases: PDGFRα(V561D), DDR1, KIT, KIT(V560G), HER4, KIT(D816E),EGFR(L858R), KIT(V654A), PDGFRα(D842V), EGFR, EGFR(L861Q),EGFR(d746-750), JAK1, RET, RET(S891A), FGFR3, ALK, WNK3, RET(Y791F),BRAF(V600E), RAF1, ROCK1, AurA, MAP2K2, RET (M918T), KDR, FGFR2, Erk1,EGFR(T790M), RET(G691S), PDGFRα(T674I), p38α, HER2, JAK3, MAP2K1, p38β,EGFR(T790M/L858R), FLT1, BRAF, KIT(T670I), MET, skMLCK, MNK1, MST1,PKD1, JAK2, YES(T348I), FGFR1, EGFR(d746-750/T790M).

Compound II showed >56 k inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK, HCK, BTK, LYNa, FRK, FYN(isoform a), FYN(isoformb), TEC, BMX, LYNb, ABL, FGR, FMS, BLK, EPHA1, ABL(E255K). Compound IIshowed <50% inhibition at 1 nM on the following recombinant kinases:PDGFRβ, PDGFRα, PIK3CA/PIK3R1, KIT, KIT(V560G), EGFR, HER2, YES(T348I),ITK, p38α, ABL(T315I), RAF1, p38β, BRAF. Compound II showedconcentration-dependent inhibition of recombinant BTK activity with anIC₅₀ of <1 nM.

Compound II showed >50% inhibition at 10 nM on the following recombinantkinases: BTK, PDGFRβ, KIT(V560G) PDGFRα, KIT. Compound II showed <50%inhibition at 10 nM on the following recombinant kinases: PIK3CA/PIK3R1EGFR, p38α, RAF1, HER2, p38β, BRAF.

Compound II showed >50% inhibition at 100 nM on the followingrecombinant kinases: PDGFRα, KIT(V560G), KIT, PDGFRβ, EGFR. Compound IIshowed <50% inhibition at 100 nM on the following recombinant kinases:p38α, RAF1, PIK3CA/PIK3R1, HER2, p38α, BRAF.

Compound IV showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).

Compound V showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).

Compound VI showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).

Compound VII showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).

Compound VIII showed >50% inhibition at 1 nM on the followingrecombinant kinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL(E255K).

Compound IX showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).

Compound X showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).Compound X showed concentration-dependent inhibition of recombinant ABL,ABL (E255K), BTK and BTK (C481S) activity with IC₅₀ values of <1 nM,respectively.

Compound XI showed >50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).Compound XI showed concentration-dependent inhibition of recombinantABL, ABL (E255K), BTK and BTK (C481S) activity with IC₅₀ values of <1nM, respectively.

Compound XII showed 50% inhibition at 1 nM on the following recombinantkinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL (E255K).

Compound XIII showed >50% inhibition at 1 nM on the followingrecombinant kinases: SRC, YES, LCK, HCK, BTK, LYNa, TEC, BMX, ABL, ABL(E255K).

Example 16 Cell Inhibition Assay

Cell lines of SU-DHL-4 (ATCC® CRL-2957™), K-562 (ATCC® CCL-243™), andMino (ATCC® CRL-3000™) were purchased from American Type CultureCollection (ATCC, Manassas, Va.). SU-DHL-4 cells and Mino cells grew inATCC-formulated RPMI-1640 medium (ATCC) supplemented with 10% fetalbovine serum (FBS)(Gibco, Life Technologies) (complete medium)a T-75flask at 37° C. under 5% CO₂ with saturated humidity. K-562 cells grewin ATCC-formulated Iscove's Modified Dulbecco's Medium (IMDM)supplemented with 10% FBS (Gibco, Life Technologies) (complete medium) aT-75 flask at 37° C. under 5% CO₂ with saturated humidity. When thecells grew to a concentration of approximately 1×10⁶ cells/mL, they werediluted to 2.5-5×10⁴ cells/mL with the corresponding complete medium foreach cell line. A 200 μL aliquot of the cell suspension was added to thewell of a 96-well plate which was pre-added with 1 μL of the testcompounds at various concentrations, and the plate was incubated at 37°C. under 5% CO₂ with saturated humidity for 48 h. At end of the cellculture, a 10 μL aliquot of PrestoBlue® Cell Viability reagent(ThermoFish Scientific) was added into the well, and the plate wasincubated at 37° C. for approximately 60 min. The absorptions at 570 and600 nm were measured with a SpectraMaxMicroplate reader (MolecularDevices). The absorbance at 570 nm was normalized to that at 600 nm. Thenormalized absorbance at 570 nm was used for IC₅₀ calculation followingthe median-effect plot method (TC Chou, Theoretical basis, experimentaldesign, and computerized simulation of synergism and antagonism in drugcombination studies. Pharmacol Rev. 2006; 58:621-681).

Compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, andXIV showed concentration-dependent inhibition of growth of SU-DHL-4 withIC₅₀ values of <15 nM, respectively.

Compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, andXIV showed concentration-dependent inhibition of growth of K562 cellswith IC₅₀ values of <2 nM, respectively.

Compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII and XIVshowed concentration-dependent inhibition of growth of Mino with IC₅₀values of <50 nM, respectively.

Example 17 Metabolic Stability

Metabolic stability in liver microsomes:test compounds and dasatinibwere incubated at a concentration of 1 μM in human liver microsomes (0.5mg/mL) (Corning Inc., Tewksbury, Mass.) in 0.1 M phosphate buffercontaining 10 mM MgCl₂, 1 mM NADPH and 2 mM UDPGA for time pointsranging from zero to 60 min at 37° C. (see FIG. 1). Dasatinib(HY10181/CS0100, 302962-49-8, Batch No: 13044) was purchased fromMedChemExpress USA (Monmouth Junction, N.J.). The incubation reactionsat various time points were quenched by adding 2× volumes ofacetonitrile containing 100 nM reserpine. The quenched incubationsamples were centrifuged at 4000 RPM for 10 min, and the supernatantsinjected for LC/MS/MS analysis. LC/MS/MS analysis was carried out on aAB Sciex 4000 Q Trap LC/MS/MS system coupled with Shimadzu ProminenceUFLCXR 20 series (including a CBM-20A controller, two LC-20ADXR solventdelivery units, SIL-20AC HT autosampler, CTO-20A column oven, and aSPD-20A UV detector). The samples were separated on a PhenomenexColumbus column (C18, 4 μm, 50×2 mm) eluted with two solvent systems: 2mM ammonium acetate in water containing 0.1% formic acid (A) andmethanol (B) at a linear gradient starting with 25% B. Electrosprayionization in positive mode was used to acquire LC/MS/MS data. The invitro t1/2 and intrinsic clearance were calculated using method previousreported (Obatch S. Prediction of human clearance of twenty-nine drugsfrom hepatic microsomal intrinsic clearance data: An examination of invitro half-life approach and nonspecific binding to microsomes. DrugMetab Dispos. 1999; 27(11):1350-9).

Example 18 Pharmacokinetics

Pharmacokinetics: Compound IV and dasatinib (2-in-1) were both dissolvedat a concentration of 0.2 mg/mL (each compound) in water containing 2.5%DMSO, 20% propylene glycol 300, and 8% dextrose solution (50%) forintravenous dosing, and at a concentration of 0.25 mg/mL (each compound)in water containing 5 DMSO, 20% propylene glycol 300, and 10% dextrosesolution (50%) for oral dosing. Compound V and compound III (2-in-1)were both dissolved at a concentration of 0.2 mg/mL (each compound) inwater containing 2.5% DMSO, 20% propylene glycol 300, and 8% dextrosesolution (50%) for intravenous dosing, and at a concentration of 0.25mg/mL (each compound) in water containing 5% DMSO 20% propylene glycol300, and 10% dextrose solution (50%) for oral dosing. Compound X anddasatinib (2-in-1) were both dissolved at a concentration of 0.2 mg/mL(each compound) in water containing 2.5% DMSO, 30% propylene glycol 300and 10% dextrose solution (50%) for intravenous dosing, and at aconcentration of 0.5 mg/mL (each compound) in water containing 5% DMSO,50% propylene glycol 300, and 10% Solutol® HS 15 (Sigma-Aldrich) fororal dosing. Compound XI and compound III (2-in-1) were both dissolvedat a concentration of 0.2 mg/mL (each compound) in water containing 2.5%DMSO, 30% propylene glycol 300, and 10% dextrose solution (50%) forintravenous dosing and at a concentration of 0.5 mg/mL (each compound)in water containing 5% DMSO, 50% propylene glycol, and 10% Solutol® HS15 for oral dosing.

Sprague-Dawley rats (approximate weight 275-300 g, N=3) were dosedintravenously at 5 mL/kg and orally at 10 mL/kg with the above dosingsolutions. Blood samples were collected into tubes containing EDTA asthe anticoagulant, at 0, 0.25 (intravenous only), 0.5, 1, 2, 4, 8, 10,and 24 h post-dosing, and plasma samples were prepared bycentrifugation. The plasma samples were mixed with 3× volumes ofacetonitrile containing 100 nM reserpine as the internal standard, andcentrifuged at 4000 RPM for 15 min. The supernatants were injected forLC/MS analysis which were carried out on a AB Sciex 4000 Q Trap LC/MS/MSsystem coupled with Shimadzu Prominence UFLCXR 20 series (including aCBM-20A controller, two LC-20ADXR solvent delivery units, SIL-20AC HTautosampler, CTO-20A column oven, and, a SPD-20A UV detector). Thesamples were separated on a Phenomenex Columbus column (C18, 4 μm, 50×2mm) eluted with two solvent systems: 2 mM ammonium acetate in watercontaining 0.1% formic acid (A) and methanol (B) at a linear gradientstarting with 25% B. Electrospray ionization in positive mode was usedto acquire LC/MS/MS data. Plasma concentrations of compounds III, IV, V,X, XI and dasatinib were quantitated using standard curves,respectively.

Example 19 Tissue Distribution

Lung Tissue and Plasma Concentration Ratios: Compound X and dasatinib(2-in-1) were both dissolved at a concentration of 0.5 mg/mL (eachcompound) in water containing 5% DMSO, and 150% propylene glycol 300,and 9% Solutol® HS 15. Compound XI and dasatinib (2-in-1) both dissolvedat a concentration of 0.5 mg/mL (each compound) in water containing 5%DMSO, 20% propylene glycol 300. [0190] CD-1 mouse (approximate weight 30g, N=3) were dosed by oral gavage at 10 mL/kg. Blood samples werecollected into tubes containing EDTA as the anticoagulant at 1, 2, 4, 10h post-dosing for Compound X and predose, 1, 2, 3, 8, 10 and 24 hpost-dosing for Compound XI. The plasma samples were prepared bycentrifugation. Lung samples were also collected at each of the abovetime points. The lung samples were homogenated in 4× distilled water(v/w). The plasma samples and the homogenated lung tissue samples weremixed with 3× volumes of acetonitrile containing 100 nM reserpine as theinternal standard, and centrifuged at 4000 RPM for 15 min. Thesupernatants were injected for LC/MS analysis which were carried out ona AB Sciex 4000 Q Trap LC/MS/MS system coupled with Shimadzu ProminenceUFLCXR 20 series (including a CBM-20A controller, two LC-20ADXR solventdelivery units, SIL-20AC HT autosampler, CTO-20A column oven, and aSPD-20A UV detector). The samples were separated on a PhenomenexColumbus column (C18, 4 μm, 50×2 mm) eluted with two solvent systems: 2mM ammonium acetate in water containing 0.1% formic acid (A) andmethanol (B) at a linear gradient starting at 25%. Electrosprayionization in positive mode was used to acquire LC/MS/MS data. Theplasma and tissue concentrations of Compounds X, XI and dasatinib weredetermined using standard curves, respectively.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of appended claim. All publications, patents, and patentapplications cited herein are hereby incorporated by reference.

I claim:
 1. A method of treating a subject suffering from a cancerselected from Philadelphia chromosome-positive (Ph+) chronic myeloidleukemia (CML), Philadelphia chromosome-positive acute lymphoblasticleukemia (Ph+ ALL), diffuse large B-cell lymphoma (DLBCL), chroniclymphocytic leukemia (CLL), follicular lymphoma, marginal zonelymphomas, mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia(WM), T-cell lymphomas, and/or multiple myeloma, comprisingadministering to the subject a therapeutically effective amount of acompound having the structure

and/or a salt, enantiomer, and enantiomeric mixture thereof.
 2. Themethod of claim 1, wherein said compound is compound X:

and/or a salt thereof.
 3. The method of claim 2, wherein the cancer isat least one of Philadelphia chromosome-positive (Ph+) chronic myeloidleukemia (CML), Philadelphia chromosome-positive acute lymphoblasticleukemia (Ph+ ALL), and diffuse large B-cell lymphoma (DLBCL).
 4. Themethod of claim 1, wherein said compound is compound XI:

and/or salt thereof.
 5. The method of claim 4, wherein the cancer is atleast one of Philadelphia chromosome-positive (Ph+) chronic myeloidleukemia (CML), Philadelphia chromosome-positive acute lymphoblasticleukemia (Ph+ ALL), and diffuse large B-cell lymphoma (DLBCL).
 6. Amethod of treating a subject suffering from a cancer selected fromPhiladelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML),Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL),diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia(CLL), follicular lymphoma, marginal zone lymphomas, mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia (WM), T-cell lymphomas,and/or multiple myeloma, comprising administering to the subject acompound having the structure of:

and/or a salt, enantiomer, and enantiomeric mixture thereof, incombination with at least one additional therapeutic agent, and apharmaceutically acceptable excipient, diluent, and/or carrier.
 7. Themethod of claim 6, wherein the at least one therapeutic agent is achemotherapeutic agent, a biologic agent, an immunosuppressive agent, asteroid hormone, and/or non-steroidal anti-inflammatory agent.
 8. Themethod of claim 6, wherein said compound is compound X:

and/or salt thereof.
 9. The method of claim 6, wherein said compound iscompound XI:

and/or salt thereof.
 10. A method of treating a subject suffering fromcancer selected from Philadelphia chromosome-positive (Ph+) chronicmyeloid leukemia (CML), Philadelphia chromosome-positive acutelymphoblastic leukemia (Ph+ ALL), and diffuse large B-cell lymphoma(DLBCL) comprising administering to the subject suffering from cancer atherapeutically effective amount of a compound having the structure

and/or salt thereof.